Recovery from swelling of hepatocytes and selected other epithelia is triggered by intracellular Ca(2+) release from the endoplasmic reticulum, which leads to fluid and electrolyte efflux through volume-sensitive K(+) and Cl(-) channels. The aim of this study was to determine the mechanisms responsible for swelling-mediated hepatocellular Ca(2+) mobilization. Swelling of HTC rat hepatoma cells, evoked by exposure to hypotonic medium, elicited transient increases in intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and cytosolic [Ca(2+)]. The latter was attenuated by inhibition of phospholipase C (PLC) with and by IP(3) receptor blockade with 2-aminoethoxydiphenyl borate, but it was unaffected by ryanodine, an inhibitor of intracellular Ca(2+)-induced Ca(2+) release channels. Hypotonic swelling was associated with a transient increase in tyrosine phosphorylation of PLCgamma, with kinetics that paralleled the increases in intracellular IP(3) levels and cytosolic [Ca(2+)]. Confocal imaging of HTC cells exposed to hypotonic medium revealed a swelling-induced association of tyrosine-phosphorylated PLCgamma with the plasma membrane. These findings suggest that activation of PLCgamma by hepatocellular swelling leads to the generation of IP(3) and stimulates discharge of Ca(2+) from the endoplasmic reticulum via activation of IP(3) receptors. By extension, these data support the concept that tyrosine phosphorylation of PLCgamma represents a critical step in adaptive responses to hepatocellular swelling.