The rat pancreatic acinar tumor cell line, AR42J, is widely used to study pancreatic acinar cell biology and biochemistry. In addition to the well-documented exocrine cell features, we have identified by immunofluorescence and by electron microscopy the co-expression of small neuroendocrine (NE) vesicles using the NE vesicle-specific markers synaptophysin and "protein S.V.2." AR24J cells store [3H]GABA, which is secreted upon potassium depolarization in a calcium-dependent manner. In addition, we found the expression of the receptor for the neurotransmitter substance P by using a receptor-specific cDNA probe. Glucocorticoid treatment, which profoundly inhibits cellular growth and induces differentiation, results in a rapid decrease of substance P receptor (SPR) gene expression as assessed by Northern blot analysis. Intracellular Ca2+ mobilization was then determined in response to substance P in control cells and glucocorticoid-pretreated cells by dual wavelength spectrophotometry using fura-2 in single cells. Glucocorticoid-mediated down-regulation of substance P receptors resulted in a dose- and time-dependent decrease of the intracellular Ca2+ mobilization stimulated by substance P. In summary, these data indicate that AR42J cells display an amphicrine phenotype with two differentially regulated secretory pathways; during glucocorticoid-induced differentiation, the cells become less sensitive to substance P stimulation as a consequence of reduced gene expression of the substance P receptor.