The objective of the present study was to examine the molecular mechanisms whereby angiotensin II (Ang II) stimulates smooth muscle (SM) α-actin expression in rat aortic smooth muscle cells (SMCs). Nuclear run-on analysis and transfection studies indicated that the effects of Ang II on SM α-actin were mediated at least in part at the transcriptional level. Transfection of various rat SM α-actin promoter/chloramphenicol acetyltransferase (CAT) constructs into SMCs demonstrated that the first 155 bp of the SM α-actin promoter was sufficient to confer maximal Ang II responsiveness, conferring an ≈4-fold increase in reporter activities in these SMCs compared with vehicle-treated SMCs. Mutation of either of two highly conserved CArG elements, designated A (−62) and B (−112), completely abolished Ang II–induced increases in reporter activity, whereas mutation of a homeodomain-like binding sequence at −145 (ATTA) reduced reporter activity by half. Results of EMSAs showed that nuclear extracts from Ang II–treated SMCs exhibited enhanced binding activity of serum response factor (SRF) to the CArG elements and of a homeo-domain factor, MHox, to the ATTA element. Northern analyses showed that Ang II also stimulated marked increases in MHox mRNA levels. Western analyses demonstrated that Ang II–induced increases in SRF binding were not due to increased SRF protein expression. Recombinant MHox markedly enhanced binding activity of SRF in EMSAs. Finally, MHox overexpression transactivated a SM α-actin promoter/CAT reporter construct by ≈3.5-fold in transient cotransfection studies. These results provide evidence for involvement of a homeodomain transcription factor, MHox, in Ang II–mediated stimulation of SM α-actin via a CArG/SRF-dependent mechanism.