Although retinoic acid (RA) has been known for many years to be a modulating agent that plays a role in generating both granulocytes and monocytes, the molecular mechanism underlying this role has not been defined in the monoblast lineage. In particular, the part played by the retinoid X receptors (RXRs), which are members of the steroid/thyroid hormone nuclear receptor family, has not been explored. In this study, therefore, the human monoblastic leukemia cell line U937 has been used as a model system to investigate the role of one of the RXRs, RXR-alpha, in monoblast differentiation. RXR-alpha mRNA was present in untreated U937 cells, and levels increased after induction of differentiation with phorbol ester. The same was found for RXR-beta mRNA. Using plasmids containing sense or antisense RXR-alpha sequences under the control of an inducible promoter, we generated stably transfected cell lines which expressed either increased or decreased levels of RXR-alpha, respectively. The sense cell lines (U alpha S and its clonal derivative alpha G2S) showed increased sensitivity to RA, while the antisense cell lines (U alpha A and its clonal derivative alpha B5A) showed decreased sensitivity to RA, as demonstrated by growth inhibition and by regulation of an RA-responsive reporter gene. Both U alpha A and alpha B5A also failed to respond to another modulating agent, 1 alpha,25-dihydroxycholecalciferol (DHCC), but only U alpha S and not alpha G2S showed an enhanced response to DHCC. The combination of RA and DHCC together inhibited growth of both sense and antisense cell lines. In addition, alpha G2S exhibited increased expression of CD11b and CD54, while alpha B5A cells showed increased expression of CD102, suggesting that RXR-alpha has a role in regulating expression of cell adhesion molecules in U937 cells. These results demonstrate that RXR-alpha has a role in mediating growth inhibition and cell adhesion during myelomonocytic differentiation, and suggest that different species of heterodimers involving RXR-alpha may control the acquisition of different features of mature monocyte/macrophage function.