Angiotensin (Ang) II-stimulated phosphorylation of signal transducer and activator transcription (STAT) 3 in rat hepatocytes and the effects of ethanol on this activation were investigated. Angiotensin II (100 nM) stimulated Tyr705 and Ser727 phosphorylation of STAT3 and formation of sis-inducing factor complexes. In the presence of U-0126 (10microM), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor, Ang II further increased Tyr705 phosphorylation of STAT3 but completely abrogated Ser727 phosphorylation of STAT3. Inhibition of p42/44MAPK also increased STAT3 DNA-binding activity. Pretreatment with ethanol (100mM) for 24h resulted in decrease in Tyr705 phosphorylation of STAT3 by ethanol alone and inhibition of Tyr705 phosphorylation of STAT3 stimulated by Ang II. Although ethanol potentiates Ang II stimulated p42/44 MAPK activation in hepatocytes, ethanol inhibited Ser727 phosphorylation of STAT3 stimulated by Ang II. Angiotensin II-stimulated STAT3-binding activity was not significantly affected by ethanol treatment. These results suggest a negative regulation of Ang II-stimulated STAT3 tyrosine phosphorylation and STAT3-binding activity through p42/44 MAPK activation in hepatocytes. However, ethanol modulation of Ang II-stimulated STAT3 phosphorylation occurs by MAPK independent mechanisms. Ethanol potentiation of MAPK signaling without suppression of STAT3 function may modulate the course of alcoholic liver injury.