Approximately 1 kilobase of genomic DNA from the chicken fast myosin light-chain 1f/3f gene 5' to the transcriptional start sites for each light-chain mRNA was sufficient for differentiation-dependent, tissue-restricted expression. This was determined in primary chick myoblast cultures transfected with the chloramphenicol acetyltransferase (CAT) expression vector p8CAT containing these 5'-flanking sequences. The expression of CAT activity from both light-chain promoters was 10- to 20-fold higher in differentiated myotubes than in fibroblasts or myoblasts grown in bromodeoxyuridine. In contrast, the beta-actin and Rous sarcoma virus promoters joined to the CAT gene were expressed equally in all cell backgrounds tested. Even though the relative timing of light-chain 1f and 3f expression was altered, tissue-restricted, differentiation-dependent expression of the light-chain mRNAs was maintained with these 5' cis-acting sequence elements.