Although methacholine (MCh) and a Ca2+ ionophore A23187 do not enhance the tissue adenosine 3',5'-cyclic monophosphate (cAMP) level by themselves, they markedly potentiate isoproterenol (ISO)-induced tissue cAMP accumulation in isolated simian eccrine sweat glands in a dose-dependent manner. The agonist concentration producing 50% of the maximal response of such a potentiated cAMP accumulation was 2.1 X 10(-7) M for MCh, 2.5 X 10(-7) M for ISO, and 2.9 X 10(-6) M for A23187. Unlike cAMP accumulation induced by ISO alone, MCh-stimulated ISO-induced cAMP accumulation is dependent on extracellular Ca2+. MCh- plus ISO-induced cAMP level is tripled by 10(-2) M theophylline (TH), and while the ISO-induced cAMP level was also elevated by TH, it was not enhanced to the level of ISO-plus MCh-induced cAMP accumulation, indicating that phosphodiesterase inhibition is not the major mechanism for the augmentative effect of MCh. The augmentative effect of MCh was seen only in the secretory portion, whereas that of A23187 was seen in both the secretory portion and the duct. The data suggest that MCh-induced augmentation of ISO-stimulated cAMP accumulation is due to increased cAMP formation, not decreased cAMP degradation, and that it may be mediated by an elevated intracellular Ca2+.