Dissociated acinar cells prepared from guinea pig and mouse pancreas were intact on the basis of structure, ion content, and their ability to increase release of amylase in response to bethanechol and the calcium ionophore A23187. Guinea pig but not mouse acinar cells increased amylase release in response to caerulein. An increase in the concentration of K+ in the medium, which increases amylase release from whole pancreas, did not increase release. The effect of varying the ionic content of the medium on basal and stimulated amylase release was studied. Bethanechol and caerulein were still able to stimulate amylase release when Ca2+ was omitted from the medium, whereas stimulation induced by A23187 was abolished. Elevation of the concentration of Mg2+ did not affect basal or stimulated amylase release. Removal of Na+ from the medium initially had no effect on amylase release although bethanechol-stimulated release by mouse cells was inion of the HCO3- or Cl- content of the medium did not affect cholinergic stimulation of secretion. It is concluded that stimulated amylase release by isolated acinar cells is relatively independent of the ionic constituency of the bathing medium.