Growth factors are known to stimulate colonic proliferation via activation of protein kinase C by production of diacylglycerol (DAG) from membrane phosphatidyl inositol. Previous studies from our laboratories have shown that fecal bacteria can produce and metabolize DAG and that DAG can be absorbed by colonocytes, and thus might contribute to neoplasia. Calcium is a putative chemopreventive agent, and we have shown that calcium administration reduces fecal DAG concentrations, as well as rectal proliferation in patients after jejuno-ileal bypass surgery. The present study in normal volunteers eating self-selected diets demonstrates that fecal DAG concentrations are very constant with a coefficient of variation from 6.7 to 10.2%. Calcium administration showed a trend to reduce fecal DAG by 11% (P < 0.08). We conclude that fecal DAG levels can be determined in individuals on a self-selected diet from a single stool determination. If the trend to reduce fecal DAG by calcium is verified in more extensive studies, then the effects of calcium used in chemopreventive might, in part, reflect changes in the luminal lipid content.