T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1alpha, and STAT1beta, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12-induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12-induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-gamma (IFN-gamma) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-gamma compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12-induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-gamma is not a direct effect of IFN-gamma on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-gamma. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-gamma production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells.