Our previous studies demonstrated roles of cyclic nucleotides in gamma-globin gene expression. We recently found that, upon activation of the cAMP pathway, expression of the gamma-globin gene is inhibited in K562 cells but induced in adult erythroblasts. Here we show that c-Myb, a proto-oncogene product that plays a role in cell growth and differentiation, is involved in the cAMP-mediated differential regulation of gamma-globin gene expression in K562 cells and primary erythroblasts. Our studies found that c-Myb is expressed at a high level in K562 cells compared to primary erythroblasts, and that c-Myb expression is further increased following the treatment with forskolin, an adenylate cyclase activator. The induction of gamma-globin gene expression was also inhibited in K562 cells by raising the levels of c-Myb expression. Importantly, forskolin-induced erythroid differentiation in K562 cells, as determined by the expression of glycophorins and CD71, suggesting that high-level expression of c-Myb may not be sufficient to inhibit the differentiation of erythroid cells. In contrast, c-Myb was not expressed in adult erythroblasts treated with forskolin and primary erythroblasts may lack the c-Myb-mediated inhibitory mechanism for gamma-globin gene expression. Together, these results show that the cAMP pathway blocks gamma-globin gene expression in K562 cells by increasing c-Myb expression and c-Myb plays a role in defining the mode of response of the gamma-globin gene to fetal hemoglobin inducers in erythroid cells.