Smooth muscle gamma-actin (SMGA) is both an early marker of smooth muscle cell differentiation, which demonstrates an expression pattern restricted to smooth muscle and the post meiotic spermatocyte. Serum response factor (SRF) DNA-binding is an important regulator of muscle differentiation, including SMGA expression during smooth muscle cell differentiation. RhoA, a low molecular weight GTPase protein, can regulate cardiac, skeletal, and smooth muscle differentiation through SRF-dependent mechanisms. This study's purpose was to examine RhoA expression during smooth muscle cell development, and determine if the SMGA promoter activity is sensitive to RhoA-mediated signaling through SRF. Additionally, the study identified the promoter regulation modifying SMGA expression by RhoA signaling. Western blot analysis of embryonic chick gizzard whole protein extracts during 5 to 14 days of development demonstrated a large induction of RhoA (10-fold) and beta1 integrin expression at day 8, which corresponds to the time SMGA expression and differentiation are occurring. Transient transfections in CV-1 fibroblast cells demonstrated that co-overexpression of SRF and RhoA could induce a 40-fold induction of -176 bp SMGA promoter activity. Mutational analysis demonstrated that serum response element (SRE)-1, but not SRE2, was necessary for RhoA/SRF activation of the SMGA promoter. Deletion analysis revealed that although SRE1 was necessary for SMGA promoter activation by RhoA and SRF, it was not sufficient, implicating a possible obligatory role of additional promoter sequences in the response. Overexpression of a mutated SRF protein that was unable to bind DNA demonstrated that the 40-fold RhoA/SRF activation was largely dependent on SRF binding to the SMGA promoter. Thus, as the SMGA promoter appears to be a target of RhoA-mediated transcriptional regulation, the uncovering of these signaling mechanisms effecting SMGA promoter activity should provide a regulatory paradigm that can then be examined during the regulation of other smooth muscle genes.