Mouse and rat genomic DNA libraries were screened by reduced stringency hybridization with the DNA-binding domain of the c/ebp gene as a probe. Three genes were isolated that encode bZIP DNA-binding proteins (designated CRP1, CRP2, and CRP3) with strong amino acid sequence similarities to the C/EBP-binding domain. CRP2 is identical to the protein described recently by other groups as NF-IL6, LAP, IL-6DBP, and AGP/EBP, whereas CRP1 and CRP3 represent novel proteins. Several lines of evidence indicate that these three proteins, along with C/EBP, comprise a functional family. Each bacterially expressed polypeptide binds to DNA as a dimer with recognition properties that are virtually identical to those of C/EBP. Every member also bears a conserved cysteine residue at or near the carboxyl terminus, immediately following the leucine zipper, that at least in vitro allows efficient disulfide cross-linking between paired zipper helices. We developed a gel assay for covalent dimers to assess leucine zipper specificities among the family members. The results demonstrate that all pairwise combinations of dimer interactions are possible. To the extent that we have examined them, the same heterodimeric complexes can be detected intracellularly following cotransfection of the appropriate pair of genes into recipient cells. All members are also capable of activating in vivo transcription from promoters that contain a C/EBP-binding site. Our findings indicate that a set of potentially interacting C/EBP-like proteins exists, whose complexity is comparable to that of other bZIP protein subfamilies such as Jun, Fos, and ATF/CREB.