A normal level of interferon (IFN) responsiveness via the Stat1 transcription factor is critical to the host, since decreased Stat1 signaling causes immune compromise and increased signaling is associated with inflammatory and neoplastic disease. Here we report how this balance may be influenced by novel alterations in the efficiency of Stat1 signaling. To enable disulfide-dependent and spontaneous formation of active Stat1 homodimer (as was done previously for Stat3), we engineered Stat1-CC with double-cysteine substitutions in the Src homology 2 (SH2)-homodimerization domain (at Ala-656 and Asn-658). In this case, however, mutant and wild-type Stat1 exhibited no difference in spontaneous dimerization. Moreover, Stat1-CC still required ligand-dependent Tyr-701 phosphorylation for function and exhibited hyperresponsiveness to IFN-β (that depends on Stat1/Stat2 heterodimerization) as well as IFN-γ (that depends on Stat1/Stat1 homodimerization). Hyperresponsivenss of Stat1-CC was accompanied by increased capacities for Tyr-701 phosphorylation and DNA binding, but these features were also found in a similarly substituted serine mutant (Stat1-SS) that showed no hyperresponsiveness to IFN-γ. This finding raised the possibility that SH2 domain mutations also influence downstream transcriptional efficiency. Indeed, each of these mutations also enhanced recruitment of the normally rate-limiting p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex in proportion to the level of IFN-driven transactivation and gene expression. Additional modifications indicated that the mutant residues in the SH2 domain appeared to cooperate with Ser-727 in the C-terminal domain to regulate p300/CBP interaction with Stat1. The profile of IFN responsiveness translated into the same progressive increase in the level of viral clearance from Stat1- to Stat1-SS- to Stat1-CC-expressing cells. Thus, SH2 domain determinants may be modified to direct better Stat1 phosphorylation, DNA binding, and coactivator recruitment to fully improve IFN efficacy.