The metabolism of [14C]arachidonic acid in whole homogenates of gastric corpus mucosa in the guinea pig was characterized. Identity of labeled products was validated by comigration against standards in multiple chromatographic systems and by selective synthesis inhibition. At near-physiological conditions, homogenates converted arachidonic acid to the cyclooxygenase products prostaglandins F2, E2, and D2, 6-keto-prostaglandin F1 alpha, and thromboxane B2 (in descending order of magnitude) and to a lipoxygenase product cochromatographing with 12-hydroxyeicosatetraenoic acid. However, dramatic qualitative and quantitative changes in this product profile were noted with relatively small changes in incubation temperature, substrate concentration, and especially pH. Whole homogenates generated more prostaglandin and in different proportions than did the microsomal fraction from an equal mass of mucosa. No catabolism of prostaglandin products was observed in whole homogenates. Tissue preparation and storage also influenced prostaglandin synthesis activity. These in vitro observations may have important implications for future studies of prostaglandin generation in small gastric mucosal biopsies.