SM22alpha is a putative calcium-binding protein that is expressed in cardiac, smooth, and skeletal muscle lineages during mouse embryogenesis and in adult smooth muscle cells (SMC). To define the mechanisms that regulate smooth muscle-specific gene transcription, we isolated the SM22alpha gene and analyzed its 5'-flanking region for elements that direct smooth muscle expression in transgenic mice. Using a series of promoter deletions, a region of the SM22alpha promoter containing 445 base pairs of 5'-flanking sequence was found to be sufficient to direct the specific expression of a lacZ transgene in mouse embryos in the vascular smooth, cardiac, and skeletal muscle lineages in a temporospatial pattern similar to the endogenous SM22alpha gene. However, in contrast to the endogenous gene, transgene expression was not detected in venous, nor visceral SMCs. This SM22alpha-lacZ transgene was therefore able to distinguish between the transcriptional regulatory programs that control gene expression in vascular and visceral SMCs and revealed heretofore unrecognized differences between these SMC types. These results suggest that distinct transcriptional regulation programs control muscle gene expression in vascular and visceral SMCs.