Omeprazole and pantoprazole are known to be irreversible, SH-acting inhibitors of gastric H+,K+-adenosine triphosphatase (H+,K+-ATPase). Both drugs concentration-dependently and pH-dependently inhibited K+-dependent p-nitrophenyl phosphatase (K+-pNPPase) activity in purified rabbit gastric microsomes. The potency of omeprazole was about three times that of pantoprazole in the pH ranges tested. Both drugs also inhibited acid secretion, as determined by [14C]aminopyrine accumulation in isolated rabbit gastric glands, with the potency ratio being about 5 (omeprazole over that of pantoprazole). Under conditions in which acid secretion was inhibited completely by the drugs, the total K+-pNPPase activity in the digitonin-permeabilized glands was scarcely reduced, showing an apparent discrepancy between the acid secretion and the proton pump activity. The isolated glands were stimulated with secretagogues for 30 min in the presence of the inhibitors, homogenized, and then separated into fractions in which K+-pNPPase activity was measured. Omeprazole exclusively inhibited the activity in the low-speed fraction, which was rich in the apical membranes, whereas pantoprazole did not inhibit activity in any fraction. When the time of treatment with the inhibitors was increased up to 5 hr, the inhibition of the total K+-pNPPase activity in the glands reached a plateau at an inhibition rate lower than 50% within 2 hr. This suggested that no continuous recycling of the proton pump was occurring during stimulation. The inhibitory effect of both drugs on the permeabilized gland preparation was less potent than that on the purified enzyme, especially at the higher pH, and it appeared to be partially reversible. The extent of the reduction in potency was more prominent for pantoprazole. It is concluded that a lower amount of proton pump activity needs to be inhibited by pantoprazole than by omeprazole to achieve the same extent of acid secretion inhibition. This appears to be due to the nature of pantoprazole, i.e. the requirement of low pH for activation and the partial reversibility of the inhibition.