Two 3 x 3 factorial experiments were conducted to examine the effects of dietary protein (8, 16, and 32% of energy from casein) and dietary fat (12, 24, and 48% of energy from corn oil) on the initiation and promotion of azoxymethane-induced carcinogenesis in rats. For the initiation study, 33 weanling male Sprague-Dawley rats were randomized to each of nine diets fed ad libitum. Azoxymethane was administered s.c. between the fourth to sixth weeks of feeding, providing a total dose of 6 mg/100 g body weight. All rats were subsequently fed a common diet containing 16% energy from protein and 24% energy from fat for an additional 30 to 38 weeks. For the promotion study, all rats were fed a common diet containing 16% of energy from protein and 12% of energy from fat until the completion of azoxymethane administration, when 33 rats were randomized to each of nine diets varying in fat and protein content and fed these diets until sacrifice. Low-protein diets during the initiation phase were associated with increased risk of renal adenocarcinomas (P less than 0.001) and mesenchymal (P = 0.005) malignancies. No other statistically significant relationships were found between the levels of dietary fat or protein and the prevalence of malignant lesions of the small intestine, colon, or kidney in either the initiation or promotion study (although polypoid adenocarcinoma of the colon increased suggestively from 13 to 19 to 26% of rats with increasing dietary protein during initiation). Results of a multiple logistic regression analysis, combining both studies, showed that ad libitum energy intake was significantly associated with intestinal carcinogenesis. The odds of finding an intestinal adenocarcinoma increased by 6.2 +/- 2.6% (SE) for each additional kilocalorie of mean daily ad libitum intake (P = 0.014). The quintile of rats which consumed the least averaged 60 kcal/day, while the most voracious quintile averaged 74 kcal/day. This 14 kcal/day difference in mean ad libitum intake corresponded to more than a doubling (146% increase) of the odds of developing an intestinal adenocarcinoma. These studies suggest that ad libitum energy intake is a critical factor modulating experimental colon carcinogenesis.