Regulation of the actin multigene family involves the recognition of regulatory sequences that specify the tissue type and developmental program of expression for each actin isotype. In order to investigate the underlying regulatory mechanisms, the human skeletal alpha-actin gene and its 5' regulatory region have been cloned and sequenced. This actin gene has seven exons; there is one large intron in the 5' untranslated region which is characteristic of the actins and many muscle-specific genes. The 5' flanking sequences are sufficient to direct tissue-specific and differentiation-regulated expression when transfected into the heterologous rat L8 myogenic cells, indicating a highly conserved regulatory system. The DNA sequence was compared to that of other actin genes, and several regions of sequence similarity were identified, particularly within regions known to be important for gene expression. Most notable among the conserved sequences are the CC(A/T rich)6GG (CArG box) motifs which have demonstrated interactions with trans-acting transcriptional factors. This same motif has been identified in several other genes and in some also serves as a binding site for transcription regulatory factors.