The relative change in in vitro discharge of amylase (or total protein) versus pulse-labeled protein from rat pancreatic fragments was studied after in vivo stimulation with bethanechol or saline injections. Bethanechol was injection were pulse-labeled for 5 min with [14C]L-phenylalanine. The pulse was chased for 60 min. In fragments derived from animals injected with saline, ratio of amylase (or total protein) to new secretory protein decreased at 5 and 15 min but there was no change at 45 min of in vitro incubation with 5 X 10-5 M bethanechol. In fragments derived from animals injected with bethanechol, ratio of amylase to radiolabeled secretory protein decreased at 5 and 15 min and the ratio of total protein to radiolabeled secretory protein secretion decreased at 5 but not at 15 and 45 min. There was a lack of parallelism between the ratios of amylase to radiolabeled protein secretion and total protein secretion to radiolabeled protein secretion in animals injected with bethanechol in vivo followed by in vitro incubations. These experiments indicate that the older proteins are preferentially secreted under nonstimulated conditions and mixing of granules occurs following stimulation. The time required for mixing varies depending on whether the cells are stimulated or not. These data provide support for the concept that there may be more than one subpopulation of zymogen granules in the acinar cell leading to nonparallel enzyme secretion.