Human type I interferons (IFNs) play an important role in the regulation of antiviral defense mechanisms, immunomodulatory activities, and growth control. Recent efforts have demonstrated the importance of IFNs in the activation of signal transducers and activators of transcription (STATs). The role of STAT1 and STAT2 in IFN-dependent JAK-STAT signaling is well established; however, the role of STAT3 and its activation by IFNs remains unclear. Understanding the IFN-dependent regulation of STAT3 is of increasing interest because recent studies have demonstrated that STAT3 may play a role in cancer. Studies have revealed that STAT3 is constitutively active in a number of cancer cell lines and that overexpression of an active form of STAT3 transforms normal fibroblasts. Therefore, STAT3 exhibits properties indicative of known oncogenes. In this report, we define the role of the type I IFN receptor in STAT3 activation and identify for the first time tyrosine residues present in the cytoplasmic domain of IFNAR2c that are critical for STAT3 activation. The regulation of STAT3 activation by IFNs was measured in a human lung fibrosarcoma cell line lacking IFNAR2c but stably expressing various IFNAR2c tyrosine mutants. We show here that in addition to IFN-dependent tyrosine phosphorylation of STAT3, activation using a STAT3-dependent electrophoretic mobility shift assay and a STAT3-specific reporter can also be demonstrated. Furthermore, we demonstrate that type I IFN-dependent activation of STAT3 proceeds through a novel mechanism that is dependent on two tyrosines, Tyr(337) and Tyr(512), present in IFNAR2c and contained within a conserved six-amino acid residue motif, GxGYxM. Surprisingly, both tyrosines were previously shown to be required for type I IFN-dependent STAT1 and STAT2 activation. Our results reveal that type I IFNs activate multiple STATs via the overlapping usage of two tyrosine residues located in the cytoplasmic domain of IFNAR2c.