Isolated gastric glands from rabbits and parietal cells from dogs have recently become useful in studying the control and enzymatic mechanisms of gastric H+ secretion. The present studies were performed to determine the experimental variables that account for widely differing aminopyrine accumulation reported in various publications. We found that two principle factors were responsible for wide differences in aminopyrine accumulation. First, we found that commercially available aminopyrine contained an unidentified impurity that increased with storage. A procedure for purification is included. The contaminant is not accumulated in secreting gastric glands and thereby reduces the aminopyrine ratio that can be achieved. Mixing of glands appeared to be the second important variable. It was found that incubation in 1.5-ml capped conical polypropylene centrifuge tubes in the horizontal position with shaking in the long axis of the tubes gave aminopyrine ratios that were more than double the results obtained by other mixing techniques. In addition a gland density of 1--2 mg dry wt/ml glands and a mixing rate of 110 cycles/min gave the best results. Calculations indicate that, at high gland densities, even modest amounts of impurity in the aminopyrine will significantly reduce aminopyrine ratios. With optimal conditions our greatest aminopyrine ratio was 1,050, which suggests an H+ concentration of approximately 67 mM in the canaliculi and tubulovesicular membrane system of the parietal cell. Such a level of function approaches that of the intact in vivo organ.