Recent evidence suggests that c-jun plays a pivotal role in macrophage differentiation induced by multiple agents and that cJun overexpression induces partial macrophage differentiation in the leukemic cell line U-937. The novel differentiating agent bufalin, a Na+/K+ATPase inhibitor, has also been shown to induce macrophage differentiation in U-937. In order to further define the role of c-jun in macrophage differentiation, we examined the function of c-jun/AP- 1 during bufalin induced differentiation in both c- jun transfected and parental U-937 cells. In contrast to phorbol esters, bufalin does not significantly stimulate c-jun or c-fos mRNA expression or AP- 1 transactivation. However, bufalin treatment leads to markedly greater morphologic and functional changes indicative of more extensive differentiation in the cJun overexpressing cells compared to the non- transfected controls. Furthermore, c Jun overexpressing cells maintain greater viability in the presence of bufalin and arrest in a different phase of the cell cycle than do control cell lines (G0/G1 versus S/G2M, respectively), although some apoptosis occurs in all cell lines treated with bufalin. These data suggest that while bufalin can induce some degree of differentiation in U-937 cells independent of c-jun/AP-1 controlled pathways, the involvement of these pathways by enforced cJun expression enhances the extent of differentiation and shifts the balance between differentiation and apoptosis.