Primary cultures of hepatocytes from postnatal Sprague‐Dawley rats were grown in arginine‐deficient, ornithine‐supplemented medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. This system of primary cultures of rat hepatocytes was utilized to evaluate the cytotoxicity of certain tricyclic antidepressant drugs (TCAs). The compounds tested were chosen to represent two distinct chemical classifications of TCAs: the dibenzazepine derivatives, imipramine (I) and desipramine (D), and the dibenzocycloheptadiene derivatives, amitriptyline (A) and nortriptyline (N). The study also allowed direct comparison of the parent tertiary amines, A and I, and their respective demethylated pharmacologically active metabolites, N and D. The hepatotoxicity of the compounds was determined by measuring leakage of cytoplasmic enzymes, lactate dehydrogenase (LDH) and glutamic‐pyruvic transaminase (GPT), into the culture medium and by assessing cell viability by the trypan blue dye exclusion test LDH leakage was a more sensitive index of early cellular injury in this study. The compounds demonstrated a dose‐ and time‐dependent order of toxicity; their hepatotoxic potency was ranked as A=N > D > I.