Spleen cells collected from allogeneic chimeras early after bone marrow transplantation (BMT) consistently showed suppressed proliferative responses to interleukin-2in vitroand failed to proliferate in mixed lymphocyte reaction (MLR) assays. However, isolation of Thy 1.2+T cells from the heterogeneous spleen cell suspension prior to culture resulted in heightened proliferation, suggesting the presence of cells capable of suppressing T cell responsesin vitro.When separated into subpopulations by negative and positive selection with specific monoclonal antibodies, a non-T, non-B population with immunosuppressive properties was identified. The suppressive cells were found in the spleens of both allogeneic and syngeneic chimeras, but not normal donor mice. Suppressor activity was transient and typically declined by 3 weeks post-BMT. The cells suppressed the response of alloactivated T cells isolated from BMT chimeras as well as naive donor T cells in MLR assays in a dose-dependent manner. To explore the mechanism(s) involved in the suppression, the effects of interferon-γ (IFN-γ)-specific mAb and the nitric oxide (NO) synthase inhibitorNG-methyl-l-arginine were examined. The results support a role for both IFN-γ and NO in the suppressive activity. Separation of cells based on Mac-1 expression indicated that there were both Mac-1-enriched and Mac-1-depleted cells capable of producing NO, but that the Mac-1-depleted cells were the most potent suppressors in MLR assays. The Mac-1-depleted cells still contained a residual population of Mac-1dimcells which showed increased levels of Mac-1 expression after overnight culture. Intracellular staining with an inducible nitric oxide synthase (iNOS)-specific mAb indicated that the NO-producing cells expressed the cell surface markers Mac-1 and Sca-1. When iNOS knockout transgenic mice were used as transplant donors,in vitrosuppression of T cell responses was reduced but not eliminated, suggesting that other mechanism(s) could contribute to the suppression. Collectively, these results demonstrate that Sca-1+/Mac-1+cells capable of producing NO are present in the spleens of recipients early after BMT and suggest that these cells may have immunoregulatory rolesin vivo.