As a first step in the analysis of the molecular function of the nuclear c-fos proto-oncogene product we have studied its subnuclear localization in serum-stimulated mouse fibroblasts where it forms a non-covalent, apparently monodisperse complex with another nuclear protein, p39. The c-fos/p39 complex is almost quantitatively released from intact nuclei by DNasel or micrococcus nuclease treatment under conditions where only a minor fraction of DNA and nuclear proteins is released. In gel filtration experiments, c-fos/p39 comigrates with chromatin and seems to be associated with regions of increased DNasel accessibility. c-fos/p39 is bound to chromatin by electrostatic forces of moderate strength since greater than 90% of the complex can be eluted from nuclei at 0.4 M NaCl. In vitro, the c-fos/p39 complex in nuclear extracts binds to double- and single-stranded calf thymus DNA, suggesting that the association of c-fos/p39 with chromatin is at least in part due to its interaction with DNA. In agreement with this conclusion, c-fos/p39 is released from nuclei by incubation with tRNA, presumably due to competition for binding sites. Our observations are compatible with the hypothesis that c-fos may play a role in the regulation of gene expression.