To identify the target genes modulated by the myb gene product (Myb), a co-transfection assay with a Myb expression plasmid was performed. Both c-Myb and B-Myb, another member of the myb gene family, trans-activated the human c-myc promoter. DNAase I footprint analysis using the bacterially expressed c-Myb, identified multiple c-Myb binding sites in the c-myc promoter region. Deletion analysis of the c-myc promoter suggested that some number of Myb binding sites, not a specific Myb binding site, is important for the c-Myb-induced trans-activation of the c-myc promoter. Using the c-myc-chloramphenicol acetyltransferase (CAT) construct as a reporter in a co-transfection assay, the domains of c-Myb required for trans-activation were examined. The functional domains of c-Myb identified using the c-myc promoter were almost the same as those identified previously with the artificial target gene containing Myb binding sites, but unlike the case with the artificial target gene the N-terminal half of the previously identified negative regulatory domains and the C-terminal 136 amino acids were required for the maximal trans-activation of the c-myc promoter. These results indicate that there are some differences in the regulation of Myb-dependent trans-activation in different target genes.