Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL+/+ and transgenic (PRL-/-) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL -/- mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL+/+ mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL+/+ mice. Splenocyte proliferation in male PRL -/- mice was significantly lower than in PRL+/+ mice after T-H. Marginal differences between PRL+/+ and PRL-/- mice were observed in the release of IL-2 and IFN-γ by splenocytes, while the release of IL-10 was significantly higher in PRL-/- than in PRL +/+ mice. A significant observation of our study is the release of a ∼25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL+/+ and PRL-/- mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL-/- transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice. Copyright © 2005 the American Physiological Society.