Interferon-α (IFN-α) can regulate the expression of the c-fos proto-oncogene in different cell types. Here we show IFN-α-activated binding of murine and human fibroblast nuclear factors to a DNA sequence element located in the 5′ upstream region (nucleotides −351/−337) of the c-fos gene. This element, like the conserved enhancer element, the IFN-stimulated response element (ISRE), that mediates transcriptional induction of IFN-α-inducible genes, also binds factors in response to platelet-derived growth factor (PDGF) and v-sis-conditioned medium (SCM). The IFN-inducible ISRE shares an 8-bp stretch of sequence homology with the IFN-responsive c-fos SCM element, and competes efficiently for binding of factors to the SCM. Protein-DNA cross-linking experiments with the SCM binding site identified an IFN-modulated nuclear protein of approximately 98 kD. This protein does not appear to be involved in transcription activation, since IFN-α failed to stimulate c-fos transcription in nuclear run-off assays, or the c-fos promoter in transient transcription assays of 3T3 fibroblasts. Our data nonetheless suggest the c-fos promoter may be an early target for signal transduction triggered by IFNα–receptor interaction.