We have previously described an alternatively spliced cDNA clone of the human c-myb proto-oncogene which has been shown to enhance the differentiation of Friend murine erythroleukemia cells. This clone, pMbm-2, contains unique 5' sequences which replace exon 1. The human c-myb intron 1 was sequenced to determine the exact position of this unique sequence and to further characterize the role of intron 1 in the regulation of the human c-myb gene. Here we report that intron 1 of c-myb is highly conserved between human and mouse throughout the intron, while only those sequences directly adjacent to exons 1 and 2 are conserved between human and chicken. The unique sequence of pMbm-2 was located directly adjacent to exon 2, suggesting that it arose as a product of alternative transcription initiation within intron 1. RNAase protection analysis was used to map a cluster of transcription start sites at the 5' end of exon 2. Levels of messages utilizing these start sites are proportional to those arising from the primary promoter. Functional characterization of this region revealed that this region can function as a promoter. Deletion studies have revealed the presence of negative and positive regulatory elements within this region which are utilized with different efficiencies in different cell lines. These studies suggest that cis or trans factors acting in this region may serve a dual function in both attenuation and transcription initiation.