Nuclear proteins isolated from untreated lymphoid cells formed complexes with the interferon-inducible transcriptional enhancer (IITE) containing a 73- and an 84-kDa protein, whereas the nuclear proteins of untreated myeloid cells formed complexes with the IITE which contained 50-, 65-, and 73-, but not 84-kDa nuclear proteins. The difference in the molecular masses of the nuclear proteins binding to the IITE in lymphoid and myeloid cells was due to a phosphatase present in the cytoplasm of the myeloid cells. Induction of transcriptional activation by interferon was accompanied by the binding of a 95-kDa nuclear protein to the IITE 1-4 h after the start of exposure to interferon. Cycloheximide did not inhibit the binding of the 95-kDa nuclear protein or the transcriptional activation of alpha-interferon-inducible genes. These data suggest that the induction of gene transcription by alpha-interferon in hematopoietic cells may be associated with post-translational changes in a 95-kDa nuclear protein that binds to IITE, thereby leading to transcriptional activation.