We have isolated a full-length mouse B-myb cDNA clone and used this to examine cell cycle-regulated expression of this gene. Mouse B-Myb was predicted to comprise 704 amino acids and to be 84% homologous with human B-Myb. There were three regions of extensive amino acid homology which may indicate functional domains: the first corresponded to the c-Myb DNA-binding domain, while the second had no counterpart in c-Myb but was instead homologous to a short segment of the related A-myb protein. The third region of homology is partially conserved in both c-Myb and A-Myb and may correspond to the c-Myb negative regulatory domain. Stimulation of quiescent 3T3 fibroblasts with serum was found to result in induction of B-myb expression in late G1 and to lead to high levels of gene transcripts that persisted through S phase. Similarly, maximum B-myb mRNA levels were reached in G2/M synchronized cells prior to entry into S phase. These results are consistent with a role in G1/S transition as has been suggested for c-myb.