The structures of a cDNA and the 5' upstream region of the human smooth muscle alpha-actin gene have been characterized. Transcriptional start sites and the non-coding first exon were mapped by primer extension analysis and by comparing cDNA and genomic sequences. The deduced human smooth muscle alpha-actin protein sequence is identical to the corresponding bovine protein sequence, and thus confirms that the previously determined human genomic sequence contained a mutation at codon 312. Human smooth muscle cells express only a single, 1.4-kilobase smooth muscle alpha-actin transcript. 5' Noncoding sequences that have the greatest similarity to the chicken gene are located in five noncontiguous segments, extending from approximately 250 base pairs upstream of the cap site through the first exon. Conserved sequences encompass a region required for expression and tissue-specific regulation of chicken smooth muscle alpha-actin and therefore are probably also important for expression of the human gene.