We have studied receptor-mediated Ca2+ mobilization in an established exocrine epithelial cell line (HSG-PA) derived from a human submandibular gland. These cells possess a single class of high-affinity muscarinic cholinergic receptors identified using [3H]-quinuclidinyl-benzilate (Kd = 0.17 +/- 0.07 nmol/l; Bmax = 37 +/- 2 fmol/mg protein; n = 3). The muscarinic agonist carbachol elicits a concentration dependent increase of [3H]-inositol trisphosphate in HSG-PA cells (100 mumol/l; greater than 2 fold by 30 s). Carbachol also results in a rapid, approximately 5-fold increase in cytosolic [Ca2+]. This response is made up of two components, one arising from the release of intracellular Ca2+ (La3+ insensitive; independent of extracellular [Ca2+]), the other from the entry of extracellular Ca2+ (La3+ sensitive; dependent on extracellular [Ca2+]). These Ca2+ mobilizing mechanisms are completely blocked by the muscarinic antagonist atropine (10 mumol/l) but unaffected by several voltage-dependent Ca2+ channel antagonists (verapamil, nifedipine, diltiazem) and by membrane depolarization (incubation in 55 mmol/l KCl). These results demonstrate that HSG-PA cells respond to muscarinic stimulation by mobilizing Ca2+ from an intracellular store and via a receptor-operated Ca2+ entry pathway.