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 following 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 hormones beneficial effect. In vitro lympho-proliferation assay with Nb2 cells showed complete absence of PRL in circulation of the transgenic PRL^-/- mice of both sexes, where as very high levels of the hormone were detected in the wild 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 compared to PRL^+/+ mice after T-H. Marginal differences were observed in the release of IL-2 and IFN- by splenocytes between PRL^+/+ and PRL-^/- mice, while the release of IL-10 was significantly higher in PRL^-/- than in PRL^+/+. A significant observation of our study is the release of a ~25 kDa protein in the ConA stimulated-splenocytes of male PRL^+/+ and PRL^-/- mice, which was active in the in vitro lympho-proliferation assay with Nb2 cells. It is unlikely that this protein is PRL since it is also present in the splenocyte extracts of PRL^-/- transgenic mice. Nonetheless, since control of lymphoid cell proliferation is considered as one of the characteristics of the immune system, the local release of this protein may be of significance in the differences observed in the splenocyte cytokine releases following T-H in the wild as well as the transgenic mice.