-Toxin-permeabilized gastric glands represent a functional model in which acid secretion can be elicited by either adenosine 3',5'-cyclic monophosphate (cAMP) or ATP, with proven morphological and functional transition between resting and secretory states [X. Yao, S. M. Karam, M. Ramilo, Q. Rong, A. Thibodeau, and J. G. Forte. Am. J. Physiol. 271 (Cell Physiol. 40): C61-C73, 1996.] In this study we use -toxin-permeabilized rabbit gastric glands to study energy metabolism and the interplay between nucleotides to support acid secretion, as indicated by the accumulation of aminopyrine (AP). When permeabilized glands were treated with a phosphodiesterase inhibitor, the secretory response to cAMP was inhibited, whereas the secretory response to ATP was potentiated. This implied that 1) ATP provided support not only as an energy source but also as substrate for adenylate cyclase, 2) activation of acid secretion by cAMP needed ATP, and 3) ATP and cAMP exchanged rapidly inside parietal cells. To address these issues, we tested the action of adenine nucleotides in the presence and absence of oxidizable substrates. All adenine nucleotides, including AMP, ADP, ATP, and cAMP, could individually enhance the glandular AP accumulation in the presence of substrates, whereas only a high concentration of ATP (5 mM) was able to support secretory activity in substrate-free buffer. Moreover, ATP could maintain 75-80% of maximal secretory activity in phosphate-free buffer; cAMP alone could not support secretion in phosphate-free buffer. In glands and in H⁺-K⁺-adenosinetriphosphatase-rich gastric microsomes, we showed the operation of adenylate kinase, creatine kinase, and ATP/ADP exchange activities. These enzymes, together with endogenous adenylate cyclase and phosphodiesterase, provide the recycling of nucleotides essential for the viability of -toxin-permeabilized gastric glands and imply the importance of nucleotide recycling for energy metabolism in intact parietal cells.