We present evidence that the mouse c-myb proto-oncogene encodes a transcriptional trans-activator. Trans-activation was assayed by cotransfection into CV1 monkey kidney cells of a c-myb cDNA expression plasmid together with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under the control of a test promoter and enhancer. Cotransfection with the c-myb cDNA plasmid caused a 20-fold stimulation of transcription from the promoter of the mouse alpha 2(I) collagen gene linked to tandem repeats of the simian virus 40 (SV40) enhancer element. Using different promoters in combination with varying numbers of repeats of the SV40 enhancer element, it was shown that tandem repeats of the SV40 enhancer mediated the c-myb-induced activation of transcription. These results show that the mouse c-myb gene product either is itself or induces, an activator of transcription that recognizes specific sequences in the SV40 enhancer.