Purinergic agonists regulate mucin secretion in the airway epithelial cells. This study examined the effects of the apical application of purinergic agonists on Ca(2+) influx ([Ca(2+)](i)), and mucin secretion along with their underlying signaling pathway in normal human middle ear epithelial (NHMEE) cells. The apical membrane of NHMEE cells were stimulated with various purinergic agonists, including UTP, and the [Ca(2+)](i) was measured using a miniature Ussing double perfusion chamber. P2Y(2) receptor in NHMEE cells was also localized by immunohistochemistry. UTP-induced mucin secretion was quantified by an immunoblotting assay. The order of the purinergic agonist potency with respect to [Ca(2+)](i) determined in this study was ATP = UTP > 2-MeSATP > UDP >> adenosine which is consistent with that obtained from P2Y(2) receptor activation. The P2Y(2) receptor is expressed in the apical membrane of monolayered cultured NHMEE cells. Apical UTP-induced [Ca(2+)](i) was inhibited by 2-aminoethoxydiphenyl borate (2-APB) but not by ryanodine. UTP-induced mucin secretion was inhibited by a Ca(2+) chelating agent, BAPTA-AM, and was stimulated by ionomycin. UTP-induced mucin secretion was also suppressed by U73122 and 2-APB, while Calphostin C suppressed it to a small extent and PD98059 was ineffective. Caffeine also inhibited the UTP-induced [Ca(2+)](i) and mucin secretion. These results suggest that the P2Y(2) receptor is expressed in NHMEE cells, and plays a major role in modulating the [Ca(2+)](i) from the IP(3)-sensitive intracellular Ca(2+) store. UTP-induced mucin secretion in NHMEE cells is strongly dependent on Ca(2+)- and IP(3).