Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.