The effects of epidermal growth factor (EGF) on intracellular calcium ([Ca(2+)](i)) responses to the muscarinic agonist carbachol were studied in a human salivary cell line (HSY). Carbachol (10(-4) M)-stimulated [Ca(2+)](i) mobilization was inhibited by 40% after 48-h treatment with 5 x 10(-10) M EGF. EGF also reduced carbachol-induced [Ca(2+)](i) in Ca(2+)-free medium and Ca(2+) influx following repletion of extracellular Ca(2+). Under Ca(2+)-free conditions, thapsigargin, an inhibitor of Ca(2+) uptake to internal stores, induced similar [Ca(2+)](i) signals in control and EGF-treated cells, indicating that internal Ca(2+) stores were unaffected by EGF; however, in cells exposed to thapsigargin, Ca(2+) influx following Ca(2+) repletion was reduced by EGF. Muscarinic receptor density, assessed by binding of the muscarinic receptor antagonist L-[benzilic-4,4'-(3)HCN]quinuclidinyl benzilate ([(3)H]QNB), was decreased by 20% after EGF treatment. Inhibition of the carbachol response by EGF was not altered by phorbol ester-induced downregulation of protein kinase C (PKC) but was enhanced upon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the carbachol response by EGF were both blocked by the MAP kinase pathway inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca(2+) release from internal stores and also exerts a direct inhibitory action on Ca(2+) influx. A decline in muscarinic receptor density may contribute to EGF inhibition of carbachol responsiveness. The inhibitory effect of EGF is mediated by the MAP kinase pathway and is potentiated by a distinct modulatory cascade involving activation of PKC. EGF may play a physiological role in regulating muscarinic receptor-stimulated salivary secretion.