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The Myb family of transcription factors is defined by homology within the DNA binding domain and includes c-Myb, A-Myb, and B-Myb. The protein products of the mybgenes all bind the Myb-binding site (MBS) [YG(A/G)C(A/C/G)GTT(G/A)]. A-mybhas been found to display a limited pattern of expression. Here we report that bovine aortic smooth muscle cells (SMCs) express A-myb. Sequence analysis of isolated bovine A-myb cDNA clones spanning the entire coding region indicated extensive homology with the human gene, including the putative transactivation domain. Expression of A-mybwas cell cycle dependent; levels of A-mybRNA increased in the late G1-to-S phase transition following serum stimulation of serum-deprived quiescent SMC cultures and peaked in S phase. Nuclear run-on analysis revealed that an increased rate of transcription can account for most of the increase in A-mybRNA levels. Treatment of SMC cultures with 5,6-dichlorobenzimidazole riboside, a selective inhibitor of RNA polymerase II, indicated an approximate 4-h half-life for A-mybmRNA during the S phase of the cell cycle. Expression of A-mybby SMCs was stimulated by basic fibroblast growth factor, in a cell density-dependent fashion. Cotransfection of a human A-mybexpression vector activated a multimerized MBS element-driven reporter construct approximately 30-fold in SMCs. The activity of c-myband c-mycpromoters, which both contain multiple MBS elements, were similarly transactivated, approximately 30- and 50-fold, respectively, upon cotransfection with human A-myb. Lastly, A-mybRNA levels could be increased by a combination of phorbol ester plus insulin-like growth factor 1. To test the role of mybfamily members in progression through the cell cycle, we comicroinjected c-mycand mybexpression vectors into serum-deprived quiescent SMCs. The combination of c-mycand either A-mybor c-mybbut not B-mybsynergistically led to entry into S phase, whereas microinjection of any vector alone had little effect on S phase entry. Thus, these results suggest that A-mybis a potent transactivator in bovine SMCs and that its expression induces progression into S phase of the cell cycle.