The coordinate expression of CD4 and CD8 antigens defines four major subsets of adult mouse thymocytes (CD4+CD8-, CD4+CD8+, CD4-CD8+ and CD4-CD8-) that represent definite steps in the differentiative pathway of immature T-lymphocytes. Thymocytes from adult C57BL/6 mice were cultured in the presence of ST 789, an L-Arg synthetic derivative of hypoxanthine, (1 to 100 micrograms/ml) or PHA 60.1 to 1 microgram/ml), or both for 48 hours, and then stained with monoclonal antibodies to CD4 and CD8 antigens for dual color flow cytometry analysis. The expression of CD25, was also investigated. ST 789 was ineffective in modifying the distribution of the four thymocyte subsets and did not induce the appearance of CD25 on cortical thymocytes. PHA induced a dramatic dose-dependent decrease of the CD4+CD8+ subset that, however, was neither enhanced nor antagonized by ST 789. We also tested ST 789 for its ability to stimulate intracellular free Ca++ rise in thymocytes. The compound was ineffective in this assay. Conversely, thymocytes promptly responded to PHA stimulation. It is concluded that ST 789 has no effect on normal adult thymocyte differentiation/activation pathway.