Distinct yet overlapping sets of STAT transcription factors are activated by different cytokines. One example is the differential activation of acute phase response factor (APRF, also called Stat3) and Stat1 by interleukin 6 and interferon-gamma. Interleukin 6 activates both factors while, at least in human cells, interferon-gamma recruits only Stat1. Stat1 activation by interferon-gamma is mediated through a cytosolic tyrosine motif, Y440, of the interferon-gamma receptor. In an accompanying paper (Gerhartz, C., Heesel, B., Sasse, J., Hemmann, U., Landgraf, C., Schneider-Mergener, J., Horn, F., Heinrich, P. C., and Graeve, L. (1996) J. Biol. Chem. 271, 12991-12998), we demonstrated that two tyrosine motifs within the cytoplasmic part of the interleukin 6 signal transducer gp130 specifically mediate APRF activation while two others can recruit both APRF and Stat1. By expressing a series of Stat1/APRF domain swap mutants in COS-7 cells, we now determined which domains of Stat1 and APRF are involved in the specific recognition of phosphotyrosine motifs. Our data demonstrate that the SH2 domain is the sole determinant of specific STAT factor recruitment. Furthermore, the SH2 domain of Stat1 is able to recognize two unrelated types of phosphotyrosine motifs, one represented by the interferon-gamma receptor Y440DKPH peptide, and the other by two gp130 YXPQ motifs. By molecular modeling, we propose three-dimensional model structures of the Stat1 and APRF SH2 domains which allow us to explain the different binding preferences of these factors and to predict amino acids crucial for specific peptide recognition.