Transcription factor ISGF-3 is a multiprotein, interferon alpha-activated transcription complex consisting of a 48-kDa DNA-binding protein and two proteins termed Stats (for signal transducers and activators of transcription) that become phosphorylated on tyrosine in the cell cytoplasm, a 113-kDa and either a 91- or 84-kDa polypeptide, the latter two of which arise from differentially spliced mRNAs. Using cell lines lacking the Stat91 or Stat84 proteins, we show that mutations in several different sites in the 91-kDa protein block the interferon alpha-induced phosphorylation of the 91-kDa protein and subsequent ISGF-3 formation. Although correct tyrosine phosphorylation on residue 690 of the Stat113 protein occurs independent of the Stat91/84 protein, the Stat113 phosphoprotein by itself moves to the cell nucleus much less efficiently in the absence of phosphorylated Stat91/84 protein.