In nonstimulated rabbit gastric glands, acetylsalicylic acid (10–500 μM) and indomethacin (3–300 μM) did not significantly modify the basal rate of acid secretion, whereas diclofenac and piroxicam (10–1,000 μM each) caused a marked and dose-dependent inhibitory effect (EC50= 138 and 280 μM, respectively). In gastric glands stimulated by histamine (100 μM), diclofenac also reduced the rate of acid formation in a dose-dependent manner. In contrast, acetylsalicylic acid, indomethacin, and piroxicam exerted a biphasic effect; thus low concentrations (3–100 μM) of these three agents significantly increased the rate of histamine-stimulated acid secretion (10–20% over the corresponding control value) by a cAMP-independent mechanism, whereas higher concentrations reduced the rate of acid formation. With respect to underlying biochemical mechanisms that could mediate inhibitory effects of NSAIDs on gastric acid formation, it was observed that both diclofenac and piroxicam, but not acetylsalicylic acid or indomethacin, decreased the glandular content of ATP, inhibited hydrolytic activity of gastric gland microsomal H+-K+-ATPase, and reduced the rate of H+-K+-ATPase-dependent proton transport across microsomal membranes in a dose-dependent manner. Furthermore, diclofenac and piroxicam also significantly increased passive permeability of microsomal membranes to protons. In conclusion, our work shows that diclofenac and piroxicam cause a significant reduction in the rate of basal and histamine-stimulated acid formation in isolated rabbit gastric glands at concentrations that can be attained in the gastric lumen of patients treated with these drugs. Mechanisms involved in these inhibitory effects appear to be multifocal and include different steps of stimulus-secretion coupling.