The tumor suppressor p53 and primary response gene Egr-1 are nuclear transcription factors with regulatory roles in signal transduction pathways mediating cellular proliferation and growth arrest as well as the complex genetic programs controlling differentiation and programmed cell death. We identified a physical association between these regulatory proteins in vitro and in vivo. Recombinant p53 and Egr-1 fusion proteins complexed with in vitro translates of Egr-1 or p53, respectively, or with these respective proteins in cell lysates. This protein-protein interaction was detected in vivo by immunoprecipitation and Western blot analysis of serum-activated cellular lysates with high levels of induced Egr-1 and of human lung cancer cell lines with constitutive overexpression of Egr-1 and mutant p53. A p53 mutant at codon 154 did not bind Egr-1, while p53 proteins with point mutations at residues 156, 246, 247, and 273 associated with this zinc finger transcription factor. p53 bound full-length Egr-1 and an Egr-1 mutant with a deletion of the 5' transactivation region but did not associate with Egr-1 protein lacking an internal segment that included the first two zinc finger domains, suggesting that binding may require the presence of intact zinc finger motifs. A variant-sized Egr-1 protein expressed by lung fibroblast cell line MRC-9 was also bound by p53. The interaction of these regulatory proteins may alter multiple features of their biological activity especially with regard to the specificity of transcriptional control.