A 2-kilobase fragment from the 5'-flanking region of the human myoglobin gene extending from -2038 to +7 relative to the cap site regulates expression of a heterologous reporter gene in a cell-specific and developmentally regulated manner. Functional analyses of 5' and internal deletions indicate that sequences located between -261 and -205 are essential for muscle-specific expression in cooperation with the myoglobin core promoter. A 167-base pair fragment containing these sequences (-371 to -205) enhances expression from myoglobin core promoter elements in a manner that is independent of its orientation and position relative to the cap site. When linked to the herpes simplex virus thymidine kinase promoter, this 167-base pair fragment of the myoglobin gene also enhances expression in differentiated myotubes but not in undifferentiated myoblasts or fibroblasts. Nucleotide sequences within the region that is essential for enhancing activity (-261 to -205) are conserved in other mammalian myoglobin genes (seal, mouse) and resemble sequences within control regions of other genes that are expressed selectively in striated myocytes. These data indicate that the 5'-flanking region of the human myoglobin gene contains an enhancer-like element that is important for transcriptional activation during myocyte differentiation.