A factorial experiment was conducted to examine the effects of dietary protein (8, 16, 32% of energy from casein) and dietary fat (12, 24, 48% of energy from corn oil) on the initiation of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast carcinogenesis in rats. Forty weanling female Sprague-Dawley rats were assigned to each of nine diets fed ad libitum. After 4 wk each rat received DMBA (20 mg/kg) via gastric intubation. For an additional 22 wk after carcinogen administration all rats consumed a diet containing 16% of dietary energy from protein and 24% from fat. Dietary fat, protein and ad libitum energy consumption exhibited statistically significant effects on final tumor prevalence, but interactive effects were not found. At necropsy, rats fed corn oil at 12, 24 and 48% of energy prior to DMBA administration showed tumor prevalences of 58, 58 and 85% with 116, 153 and 231 total tumors, respectively. The data indicate a significant nonlinear effect of dietary fat. Corresponding numbers for rats fed casein at 8, 16 and 32% of energy prior to DMBA were prevalences of 79, 65 and 59%, with total tumor counts of 194, 144 and 162. Higher dietary protein during the initiation phase was associated with a significant reduction in tumor prevalence, which was most striking between 8 and 16% of energy from protein. In addition, results of multiple logistic regression showed that tumorigenesis was increased with greater ad libitum energy intake. The odds of a tumor at necropsy were multiplied by 1.19 for each kilocalorie increase in ad libitum energy intake averaged over the post-DMBA phase of the experiment. An additional six weanling rats fed each diet for 4 wk were killed for assay of hepatic carcinogen metabolizing enzymes at the time corresponding to DMBA administration in the initiation experiment. Both protein and fat showed independent effects on the activity of several enzymes. However, enzyme activity did not suggest a unifying mechanism whereby these nutrients influence DMBA-induced mammary carcinogenesis.