The c-rel proto-oncogene encodes a 75-kDa protein (p75c-rel) which is present in the cytosol of chick embryo fibroblasts (CEF) associated with a distinct set of cellular proteins with molecular masses of 40, 115, and 124 kDa. CEF cultures arrested in S phase of the cell cycle, or enriched for G2 or mitotic cells, were examined to determine whether the expression of c-rel was altered during the cell cycle. Levels of p75c-rel remained constant in all portions of the cell cycle examined; however, a Rel-related protein with an apparent molecular mass of 64 kDa was detected in nuclei of S-phase cells. As cells enter G2, the level of this protein in the nucleus decreases. This protein reacts with antiserum generated against the carboxy terminus of p75c-rel in radioimmunoprecipitations and Western immunoblot experiments and was also detected in a Western immunoblot with antiserum generated against the first 161 amino acids of pp59v-rel. Thus, unlike other Rel/NF-kappa B family members, p64 has carboxy-terminal homology with c-Rel. The majority of peptides generated by partial proteolytic cleavage of p64 are shared with peptides generated by digestion of p75c-rel and/or pp59v-rel. We suggest that this protein represents a new member of the Rel family of transcription factors and is located in the nucleus of avian fibroblasts during S phase of the cell cycle.