The protein products of the fos (Fos) and jun (Jun) proto-oncogenes have been shown to associate with a DNA element known as the transcription factor activator protein-1 (AP-1) binding site. Jun (previously known as the Fos-binding protein p39) and Fos form a protein complex in the nucleus. To investigate the nature of the association of Fos and Jun with the AP-1 site, and to determine the role of protein complex formation in DNA-binding, we have reconstituted the protein-protein and protein-DNA interactions in vitro using Fos and Jun synthesized in reticulocyte lysates. The Fos-Jun complex formed extremely rapidly in vitro and possessed similar, though not identical, chromatographic and sedimentation properties to the complex isolated from cell extracts. Jun exhibited a low level of AP-1 binding activity; however, this was evident only at high concentrations of DNA. Fos did not bind to the AP-1 site on its own; however, it acted cooperatively with Jun to give enhanced DNA-binding activity. The increased affinity of the Fos-Jun complex for DNA resulted from a stabilization of the protein-DNA complex. These data demonstrate a cooperative interaction between the protein products of two proto-oncogenes with a DNA element involved in transcriptional regulation.