The cellular transduction pathways used by alpha 1-adrenergic and cholinergic agonists were compared in isolated acini from rat exorbital lacrimal glands. Peroxidase secretion was the index of protein secretion. Inositol phosphates were measured by anion exchange chromatography, intracellular free Ca2+ concentration ([Ca2+]i) by fluorescence methods using fura-2, cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels by protein binding radioassay, and protein kinase C (PKC) activity by [32P]ATP incorporation into exogenous substrate. Protein secretion stimulated by simultaneous addition of the alpha 1-adrenergic agonist phenylephrine and the cholinergic agonist carbachol was additive. Carbachol (10(-3) M) significantly increased the ratios of inositol phosphates to inositol during a 1- or 20-min incubation in contrast to phenylephrine (10(-5) to 10(-2) M), which did not. Phenylephrine (10(-3) M) significantly increased the [Ca2+]i by a maximum of 15 +/- 3 nM compared with carbachol (10(-4) M), which increased [Ca2+]i to a maximum of 90 +/- 14 nM. Phenylephrine (10(-4) M) did not increase cAMP levels. Phenylephrine (10(-5) to 10(-3) M) decreased cytosolic PKC activity in a concentration-dependent manner. Carbachol (10(-3) M) transiently caused a slight decrease in cytosolic PKC activity. Our results indicate that alpha 1-adrenergic and cholinergic agonists use separate and different pathways to stimulate the lacrimal gland.