Smooth muscle gamma-actin (SMGA) is an excellent marker of smooth muscle differentiation because it is essentially restricted to smooth muscle. As a first step toward unraveling the mechanisms underlying smooth muscle development and differentiation, we have examined the tissue-specific and developmental expression patterns of six constructs carrying portions of the murine SMGA gene linked to chloramphenicol acetyltransferase (CAT) in stable lines of transgenic mice. Based on the transgenic studies most, if not all, of the regulatory elements necessary for proper spatial and temporal expression of SMGA are present within a 13.7 kb segment of the SMGA gene containing 4.9 kb of upstream sequence, exon 1, intron 1, and a portion of exon 2 up to the start codon for translation. A second construct (SMGA11.6CAT) that lacks the distal 2.1 kb of upstream sequence but is otherwise identical to SMGA13.7CAT shows a similar level of smooth muscle-specific CAT activity. However, SMGA9.3CAT fusion gene containing only 571 bp of 5' flanking sequence, but otherwise identical to SMGA13.7CAT, and SMGA6.0CAT containing only the 4.9 kb upstream sequence, exon 1, and a miniintron 1 show a more than a 100-fold reduction of CAT activity in most smooth muscle-rich tissues. Furthermore, removal of most or all of intron 1 from a transgene with 571 bp of upstream sequence (SMGA2.0 CAT and SMGA0.6CAT) results in a near-complete or complete loss of activity, respectively, in all tissues. Overall, the studies suggest that upstream elements between -2.7 kb and -571 bp and elements within intron 1 are required for high levels of SMGA gene expression in an appropriate temporal-spatial fashion.