The involvement of cAMP as a second messenger for histamine-induced H+ secretion was studied in a physiologically active, in vitro preparation of piglet gastric mucosa. During the first 5--10 min of stimulation with either histamine or the cAMP phosphodiesterase inhibitor 3-isobutyl-1,4-methylxanthine (IBMX), increases (greater than or equal to 5-fold) in tissue cAMP content [(c-AMP]) were well correlated with the characteristic decrease in transepithelial resistance (R); these changes precede H+ secretion by several minutes. Control experiments indicate that, during these treatments, tissue [cAMP] is dominated by the [cAMP] of oxyntic cells alone; change in R and H+ are also related to activity of these cells alone. At the steady state (45 min), histamine and IBMX caused equivalent increases in H+ and decreases in R, but [cAMP] was markedly different in the two cases. With IBMX [cAMP] was elevated at least fivefold, whereas with histamine [cAMP] was less than or equal to 50% above resting levels. The tissue is also stimulated by exogenous additions of dibutyryl cAMP. A histamine-sensitive adenylate cyclase was present in isolated, purified oxyntic cells. The histamine sensitivity of the cyclase was very similar to that which the intact tissue exhibits for histamine-induced changes in H+ and R. The cyclase activity was blocked by cimetidine but not by promethazine. We conclude that during stimulation histamine activates a histamine (H2)-sensitive adenylate cyclase of oxyntic cells, and there is a rapid increase in cellular [cAMP] that is involved in activation of H+ transport and other associated changes of oxyntic cells. An active phosphodiesterase is responsible for reducing [cAMP] to a level much below the "peak" value. Other cellular factors (e.g. protein kinases and Ca2+-calmodulin) must also be involved in the maintenance of the stimulated state of oxyntic cells.