Interleukin-3 (IL-3)-, erythropoietin (EPO)-, and prolactin (PRL)-induced signal transduction via the JAK/STAT pathway was studied in the IL-3-dependent BAF-3 lymphoid cell line. Transfected cells expressing either the long form of the PRL receptor or the EPO receptor were used. We demonstrated that IL-3, EPO, and PRL activated a transcription factor related to the mammary transcription factor STAT5 but not to STAT1, -2, -3, or -4 as opposed to interferon gamma (IFN gamma) which activated STAT1 in the same cells. Similarly, PRL and EPO activated a STAT5-like factor (STAT5-L) in the rat Nb2 and the human UT7 cells expressing endogenous PRL and EPO receptors, respectively. The hematopoietic STAT5-L activated by IL-3, EPO, or PRL was identified as a 97-kDa tyrosine-phosphorylated protein. These results confer to STAT5 a much broader role than previously suggested.