In this study, the extracellular ATP (ATPo)-induced biochemical events were elucidated by comparing them with either the Fc epsilon RI- or Fc gamma R-induced events in the mouse mast cell line MC9. The omission of extracellular Ca2+ almost completely abolished the elevation of intracellular Ca2+ ([Ca2+]i) in the ATPo-stimulated cells, but only suppressed the second phase of the increase of [Ca2+]i in FcR-stimulated cells, thus suggesting that the ATPo-induced elevation of [Ca2+]i is totally dependent on the entry of extracellular Ca2+. Pretreatment with genistein, which inhibits protein kinases, especially protein tyrosine kinase, inhibited the FcR-triggered increase of [Ca2+]i, but not the ATPo-triggered one; however, such pretreatment did suppress both ATPo- and FcR-mediated beta-hexosaminidase release. An immunoblot analysis revealed that both ATPo and the cross-linking of FcRs led to tyrosine phosphorylation of 44- and 110-kDa proteins, which thus suggested that these tyrosine-phosphorylated proteins are involved in a modulation of the degranulation process following an elevation of [Ca2+]i. Pretreatment with PMA inhibited the FcR-induced [Ca2+]i increase, while not inhibiting the ATPo-induced one, thus suggesting that ATPo can mobilize [Ca2+]i even when protein kinase C (PKC) has already been activated. Pretreatment of calphostin C, a specific PKC inhibitor, had little effect on the ATPo-mediated beta-hexosaminidase secretion, thus indicating that the ATPo-induced degranulation is not mediated by PKC. Taken together, these results demonstrate that ATPo activates MC9 mast cells by a mechanism that is different from the activation induced by the cross-linking of FcRs.