Despite the importance of smooth muscle cell proliferation in vascular pathophysiological states, the mechanisms regulating smooth muscle cell growth and differentiation are poorly understood. Previous studies have shown that adult rabbit smooth muscles express two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are generated through alternative RNA splicing from a single smooth muscle MHC (SMHC) gene. In the present study, we isolated and characterized the rabbit SMHC gene promoter. DNA sequence analysis of the upstream region of the SMHC gene revealed several putative cis-DNA regulatory elements proximal to the transcription start site. Most notably, cis-acting regulatory elements that closely resemble CC(A/T)6GG (CArG box) and myocyte enhancer binding factor 2 (MEF-2)-type sequence motifs were found in the SMHC 5'-flanking region. In addition, six E-box motifs were found in the 5'-flanking region of the SMHC gene between -374 and -2109 base pairs from the transcription start site. A series of transient transfection assays using SMHC promoter deletion constructs indicated that a promoter fragment extending to 2266 base pairs upstream of the transcription start site has the highest reporter activity in cultured rat aortic smooth muscle cells. Gel mobility shift analyses using the MEF-2-like sequence located at -1540 revealed a specific DNA protein complex, whereas the CArG-like element located at -1275 did not show protein binding. The SMHC promoter construct, p509-CAT, which included neither the CArG- nor MEF-2-type motifs, conferred 32% of chloramphenicol acetyltransferase activity in the same cells, whereas the construct p188-CAT, which contained the minimal promoter elements (TATA box), was significantly less active (7%; 2.0-fold over background). This is the first report describing the promoter elements of a gene whose expression is restricted to smooth muscle cells.