The effect of supplemental dietary protein (casein) fed with high fat diets was investigated using the N-nitrosomethylurea-induced mammary tumor model. Isocaloric diets containing casein and corn oil at 19 and 15% (normal protein-high fat) or 33 and 15% (high protein-high fat) were fed ad libitum to Sprague-Dawley mother rats. Female offspring continued on the diet. Food consumption and growth curves were similar over the entire growth period. N-Nitrosomethylurea (50 mg/kg body weight) or saline was administered at 7 and 8 weeks of age via the tail vein. Dietary protein had no effect on serum prolactin or growth hormone throughout the estrous cycle: Prior to carcinogen administration, at 7 weeks old, proestrus at 5 p.m., serum prolactin was 231.6 +/- 141.0 (SE) ng/ml (12 rats) versus 292.2 +/- 141.0 (13 rats) for normal versus high protein diet groups, respectively. No difference was noted after carcinogen injection at 9, 13, 28, and 33 weeks of age. Similarly no effect was noted on serum growth hormone activity. Tumor latency was 7 weeks and incidence was 100% in normal protein (24 rats) and high protein (39 rats) groups 28 weeks after carcinogen treatment. The number of tumors per rat (4.38 +/- 0.37 versus 2.87 +/- 0.35, P less than 0.002) and average tumor weight (17.97 +/- 2.63 versus 9.94 +/- 2.92 g) were significantly greater in the high protein group. Study indicates that diet or carcinogen treatment did not alter hormone regulation during the estrous cycle. However, supplemental dietary protein increased the effect of high fat diets enhancing the mammary tumor burden.