Penelitian ini menyelidiki respon fisiologis, anatomis, dan morfologis tanaman tembakau (Nicotiana tabacum L.) terhadap cekaman logam berat: tembaga (Cu), kadmium (Cd), dan timbal (Pb). Uji dilakukan pada bulan Januari-April 2014. Sampel yang digunakan adalah tembakau umur 3-4 minggu yang dikecambahkan secara in vitro dan kemudian dipapar logam berat selama 14 hari. Desain penelitian yang digunakan yaitu rancangan acak lengkap dengan satu faktor: yaitu konsentrasi logam Cu (0 µM, 50 µM, 100 µM, 150 µM & 200 µM), Cd (0 µM, 50 µM, 100 µM, 200 µM & 300 µM), dan Pb (0 µM, 5 µM, 20 µM, 50 µM & 100 µM). Parameter yang digunakan antara lain: pertambahan panjang akar, pertambahan jumlah akar, akumulasi logam dalam akar, lokalisasi penimbunan dalam akar, dan warna daun. Bertambahnya konsentrasi logam menghambat pertumbuhan akar dan menyebabkan deposit logam pada jaringan akar dan gejala klorosis. Hasil uji Atomic Absorbtion Spectrosphotometry (AAS) menunjukkan semakin besar konsentrasi semakin banyak akumulasi logam pada jaringan akar. Akumulasi Cd pada konsentrasi 200 µM lebih besar dibanding pada konsentrasi 300 µM. Hal ini menunjukkan ada faktor lain selain konsentrasi seperti respon internal individu dan gangguan permeabilitas. Analisis kualitatif membuktikan bahwa cekaman Cu tidak berpengaruh signifikan terhadap warna daun, sedangkan pada cekaman Cd (100, 150 dan 200 µM) dan Pb (150 µM) daun mengalami klorosis. Secara umum pengaruh konsentrasi logam berat yang rendah seperti 50 µM Cu, 50 µM Cd, dan 5 µM Pb terhadap parameter fisiologis, anatomis, dan morfologis tidak berbeda nyata pada taraf 5 % dengan kontrol. Dengan demikian dapat disimpulkan bahwa tembakau mampu mentoleransi cekaman logam pada konsentrasi yang rendah.This research investigated physiological, anatomical, and morphological responses of tobacco (Nicotiana tabacum L.) stressed by heavy metals: copper (Cu), cadmium (Cd), and lead (Pb).The experiment was conducted from January-April 2014. The samples used are three-four weeks tobaccos germinated by in vitro then were exposed to heavy metals for 14 days. This study used completed random design with single factor: concentrations of Cu (0 µM, 50 µM, 100 µM, 150 µM & 200 µM), Cd (0 µM, 50 µM, 100 µM, 200 µM & 300 µM), and Pb (0 µM, 5 µM, 20 µM, 50 µM & 100 µM. Further, stress response is analyzed based on several parameters includes root elongation, root number, metal accumulation and localization in root, and leaf color. The increased metals had caused growth inhibition, metal deposit in root tissue, and chlorosis symptoms. Atomic Absorption Spectrosphotometry (AAS) results showed that higher concentration will cause root tissue accumulate more metals. Yet 200 µM of Cd accumulated more than 300 µM Cd, so it could be suggested that there are other factors than concentration in determining metal absorbtion such as internal individual respon and membrane permeability disturbance. In other hand,qualitative analysis proved that chlorosis was not found in Cu treatment but consistently notified in high concentrations of Cd (100 µM up to 200 µM) and Pb (100 µM). However, the influence of lower metals concentration such as 50 µM Cu, 50 µM Cd and 5 µM Pband control treatment were not significantly different at the level of 5% in physiological, anatomical, and morphological responses. Therefore, it can be concluded that tobacco was capable to tolerate low concentration of metal stress.