We studied the effects of carbachol on isoprenaline-induced secretion of 3[H]glucosamine-labeled high molecular weight protein from in vitro fragments of rat submandibular gland. The concentration-response curve for isoprenaline was determined alone and in the presence of one of two concentrations of carbachol. Isobolic interaction indices were calculated from the resulting curves. The role of extracellular calcium on this interaction was assessed by examining the effect of increasing concentrations of [Ca2+]e on secretion induced by a fixed concentration of isoprenaline alone and in the presence of carbachol. Carbachol alone caused a small, but statistically significant, protein secretion. Carbachol markedly shifted the isoprenaline concentration-response curve to the left in a dose-dependent manner. In the absence of extracellular calcium, carbachol did not increase isoprenaline-induced secretion, but secretion increased as the [Ca2+]e was increased. The carbachol-induced increase in secretion reached a maximal level at approximately 2.0 mM [Ca2+]e. We conclude that carbachol's increase of isoprenaline-induced secretion of 3[H]glucosamine-labeled high molecular weight protein is not additive, but is true potentiation. This potentiation requires extracellular calcium, and may be maximal at physiological [Ca2+]e.