The v-Myb oncogene causes monoblastic leukemia and transforms only myelomonocytic cells in culture. The v-Myb protein is nuclear and binds to specific DNA sequences. To identify genes regulated by v-Myb, we utilized primary cells transformed by a retrovirus encoding a v-Myb-estrogen receptor (ER) fusion protein. The Ets-2 gene was not expressed in v-Myb-ER transformed cells in the presence of estradiol, but was expressed within 4 h after estradiol withdrawal. The expression of Ets-2 also increased dramatically following phorbol ester-induced differentiation of the v-Myb-transformed BM2 cell line. Conversely, CRYP-alpha, encoding a transmembrane tyrosine phosphatase, was expressed in the presence but not the absence of estradiol in v-Myb-ER transformed cells. CRYP-alpha was downregulated during the phorbol ester-induced differentiation of BM2 cells. Although LIM-3 expression was estradiol-inducible in v-Myb-ER transformed monoblasts, LIM-3 was expressed neither in primary yolk sac cells transformed by unfused v-Myb nor in BM2 cells. We conclude that although v-Myb has been intensively studied as a transcriptional activator, v-Myb can repress biologically relevant genes such as Ets-2, which promotes macrophage differentiation. In addition, we have shown that some genes that are regulated by a v-Myb-ER fusion protein may not be relevant to the biological function of the unfused v-Myb protein.