It is well known that v-Src phosphorylates various substrates on tyrosine residue and associates with tyrosine-phosphorylated proteins as well as proline-rich ligands through its SH2 and SH3 domains, respectively, thereby inducing oncogenic transformation. A signal pathway from the cell surface to genes in the nucleus, the Jak/STAT (signal transducers and activators of transcription) pathway, has been shown to be involved in the signal transduction mechanism mediated by many cytokines and growth factors. Although a member of the STAT family, STAT3 has been reported to be constitutively activated in several v-Src-transformed cells, and it still remains unknown whether Jak molecules, which act upstream of STATs, are involved in the v-Src-induced activation mechanism of STAT3. In this study, we analyzed activations of both Jak and STAT molecules using v-Src-transformed HAG-1 cells derived from a human gallbladder adenocarcinoma. STAT3 was found to be constitutively activated in v-Src-transformed HAG-1 cells, but not in either non-transformed mock-transfected or activated c-H-ras-transfected HAG-1 cells, even though the other known STAT molecules are expressed. Furthermore, both Jak-2 and Tyk-2 were constitutively activated only in v-Src-transformed HAG-1 cells. Association of v-Src with either STAT3 or the Jak molecules was not observed. No change of this activation was detected by either interferon (IFN)-alpha2a or IFN-gamma, which had shown inhibitory effects on the growth of v-Src-transformed HAG-1 cells. These results raise the possibility that Jak-2 and Tyk-2 are both activated by v-Src, thereby contributing to the constitutive activation of STAT3 in the v-Src-transformed cells.