Several enzymes were investigated histochemically in the colons of normal male F344 rats in order to understand the function of different types of cells in this tissue. Serial methacrylate-embedded sections (2-4 microns) allowed the precise localizations of several enzymes including acid phosphatase, alkaline phosphatase, gamma-glutamyl transpeptidase, N-acetyl-beta-D-glucosaminidase (hexosaminidase), alpha-naphthyl butyrate esterase and 5'-nucleotidase. Sites reactive with periodic acid-Schiff were also localized. Gradients of enzyme activity were observed between caecum and rectum and/or from the luminal surfaces to the bases of the crypts for hexosaminidase, esterase and gamma-glutamyl transpeptidase. To our knowledge this is the first histochemical demonstration of gamma-glutamyl transpeptidase in normal rat colonic epithelial cells. The utilization of the methacrylate-embedding technique has revealed previously undescribed gradients of enzyme activity and has allowed the localization of enzyme activities not previously reported in normal rat colonic mucosa.