Centrifugal elutriation was adapted and analyzed as a method to separate rat gastric parietal cells from other fundic mucosal cells. Elutriated parietal cell fractions provided sufficient purity by morphological criteria, and fluorescence activated cell sorting analyses, yield, reproducibility and maintenance of functional responses. These characteristics allowed the study and comparison of the kinetics of histamine, isoproterenol, and forskolin-induced cyclic AMP and 14C-amino-pyrine accumulations in the presence of 1-methyl-3-isobutyl xanthine (IBMX) in parietal-cell-rich and parietal-cell-poor fractions. All three acid secretagogues studied produced the same maximal rate of acid secretion as judged by 14C-aminopyrine accumulation. Each secretagogue action peaked at different times and had different accumulation kinetics. For each agonist, cyclic AMP accumulation preceded secretion. However, the rate, extent, and temporal changes of cyclic AMP accumulation were independent of aminopyrine accumulation. HPLC separation and drug inhibition studies of cyclic nucleotide phosphodiesterase activities indicated the presence of multiple, high affinity (Type IV), but not lower affinity (Types I and II), enzyme forms in gastric mucosal cells. IBMX did not distinguish between the two forms, but SQ 65442 and RO 20-1724 were selective inhibitors. Inhibition constants of IBMX for phosphodiesterase hydrolysis agreed closely with its EC50 for histamine-stimulated acid secretion (2-8 microM). Elutriated parietal cells maintained their responses to selective receptor antagonists in the micromolar concentration range.