The intracellular concentration and rate of cyclic adenosine monophosphate (cAMP) synthesis, as measured by adenylate cyclase (AC) activity, were measured in dermal fibroblast cultures, colon cancer lines, and cells cultured from colonic epithelium and colonic adenomas. Dermal fibroblasts had higher AC activity and intracellular cAMP levels than the colon cancer lines (p less than 0.05). Benign colonic epithelial cultures (mucosa and adenomas) had AC levels similar to those found in dermal fibroblasts. To characterize further these observed differences, similar measurements were made in cultures incubated in cholera toxin (CT) or epidermal growth factor (EGF). CT stimulated AC activity and cAMP accumulation in both cancers and fibroblasts. EGF had no effect on AC activity in cancers or fibroblasts, and no effect on cAMP concentration in cancer, although EGF incubation did increase intracellular cAMP in fibroblasts. Dermal fibroblasts from colon cancer-prone patients had AC activity and cAMP concentration not significantly different, though greater, than fibroblasts from healthy individuals. Therefore, although the product of the oncogene associated with colon cancer has been shown to be an activator of AC in yeast, in human colon cancer, AC activity and intracellular cAMP concentration were much lower than in dermal fibroblasts. This difference was so great that AC activity and intracellular concentration of cAMP might be biochemical markers that can be used to differentiate colon cancer from benign cells in tissue culture.