Lacrimal gland protein secretion is primarily under the control of cholinergic muscarinic and alpha 1-adrenergic receptors. Cholinergic agonists are coupled to the activation of phospholipase C (PLC), which leads to the production of two second messenger molecules: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 increases the cytoplasmic concentration of calcium ([Ca2+]i), and DAG activates protein kinase C (PKC), two events that are thought to trigger protein secretion. Lacrimal gland alpha 1-adrenergic receptors are not coupled to the PLC pathway, although their activation leads to a slight increase in [Ca2+]i(3). We have also shown that unlike the cholinergic receptors, alpha 1-adrenergic receptors are not linked to the activation of phospholipase D in lacrimal gland acini. Thus the transduction pathway(s) used by the alpha 1-adrenergic receptors to trigger lacrimal gland protein secretion remains to be identified. PKC was originally described as a Ca2+ and phospholipid-dependent protein kinase activated by DAG produced by the receptor-mediated breakdown of phosphoinositides. Molecular cloning and biochemical techniques have shown that PKC is a family of closely related enzymes consisting of at least eleven different isoforms that has been divided into three categories: (1) conventional PKCs, including PKC alpha, beta I, -beta II and -gamma isoforms have a Ca2+ and DAG-dependent kinase activity; (2) novel PKCs, including PKC epsilon, -delta, -theta, -nu, and -mu isoforms, are Ca(2+)-independent and DAG-stimulated kinases; (3) atypical PKCs, including PKC zeta, and -iota/lambda isoforms, are Ca2+ and DAG-independent kinases. All PKC isoforms, except PKC mu, have a pseudosubstrate sequence in their N-terminal part that is thought to interact with the catalytic domain to keep the enzyme inactive in resting cells. In previous studies, we showed that lacrimal gland acini express three isoforms of PKC: PKC alpha -delta, and -epsilon. In the present study, we report the identification of two other PKC isoforms, namely PKC mu and -iota/lambda. We show that these isoforms are differentially located and that they translocate differentially in response to phorbol esters and cholinergic agonists. We also show that PKC isoforms differentially control lacrimal gland protein secretion and cholinergic-induced Ca2+ elevation. Part of these results has been recently published.