During recovery from intensive chemotherapy with cyclophosphamide (CTX), mice suffer a severe but transitory impairment in spleen cell proliferation to T-cell mitogens (Con A or anti-CD3 plus IL-2). Although CTX treatment reduced spleen T-cell cellularity, this cannot fully account for T-cell unresponsiveness. The results showed that CTX induces the colonization of spleen by an immature myeloid CD11b⁺Ly-6G⁺CD31⁺ population. Its presence closely correlated with the maximum inhibition of T-cell proliferation. Moreover, this suppressive activity was dependent on nitric oxide (NO) production in cultures since (1) higher amounts of nitric oxide and inducible nitric oxide synthase (iNOS) mRNA were produced in CTX spleen cells (CTX-SC) than in control splenocyte cultures and (2) NOS inhibitors greatly improved the proliferation of T lymphocytes. Nitric oxide production and suppressive activity were also dependent on endogenous interferon-γ (IFN-γ) production since anti–IFN-γ abrogated both effects. Finally, iNOS protein expression was restricted to a heterogeneous population of CD31⁺cells in which CD11b⁺Ly-6G⁺ cells were required to suppress T-cell proliferation. These results indicated that CTX might also cause immunosuppression by a mechanism involving the presence of immature myeloid cells with suppressor activity. This may have implications in clinical praxis since inappropriate immunotherapies in patients treated with intensive chemotherapy could lead to deleterious T-cell responses. (Blood. 2000;95:212-220)