The proposed intermediate steps in the relationship between a diet-dependent increase in colonic bile acids and proliferation of colonic cells were studied in rats. Male Wistar rats were fed diets supplemented with increasing amounts of steroids to increase the bile acid concentration of the colon. After 2 weeks, in vivo colonic proliferation was measured using tritiated thymidine incorporation into DNA. Luminal lytic activity was measured as lysis of erythrocytes by fecal water. To quantify hemolysis in the presence of fecal water, a method was developed which measures Fe-release using atomic absorption spectrophotometry. This method proved to be superior to the cell-counter method published earlier. Our results showed that steroid supplementation increased, in a dose-dependent manner, the total fecal and the soluble bile acid concentration as well as lytic activity of fecal water and colonic proliferation. A highly significant correlation between lytic activity of fecal water and colonic proliferation (r = 0.85, n = 24, P less than 0.001) was observed. These results indicate that the increase in colonic proliferation is mediated by diet-dependent increases in soluble colonic bile acid concentration and luminal lytic activity. This sequence of effects illustrates how diet could influence the risk for colon cancer.