The transcription factor cKrox was originally identified as a protein that bound to a negative transcription regulatory element in the murine alpha1(I) collagen promoter. We recently reported the cloning and characterization of human cKrox (hcKrox). Overexpression of hcKrox in NIH3T3 fibroblasts efficiently repressed the promoters of the fibronectin and alpha1(I) collagen genes (70-90%) in transient transfection assays and suppressed the endogenous genes in hcKrox expressing permanent cell lines. We have now isolated genomic clones and cDNAs encoding two novel transcription factors related to hcKrox termed hcKrox-beta and hcKrox-gamma (the original clone is now referred to as hcKrox-alpha). Both contain three kruppel-like zinc-finger DNA binding motifs that are 71-78% identical to those of hcKrox-alpha. The NH(2)-terminus of all three proteins contains a POZ domain, a conserved 120 amino acid motif involved in transcriptional repression and protein dimerization. RT-PCR experiments demonstrate that all three hcKrox family members are expressed in foreskin and dermal fibroblasts. Transient transfection studies in NIH3T3 fibroblasts demonstrate that hcKrox-alpha -beta and -gamma, as well as the murine cKrox-beta homologue, LRF, suppress transcription driven by promoters for the alpha1(I) and alpha2(I) collagen, fibronectin and elastin genes. Electrophoretic mobility shift assays and coimmunoprecipitation studies suggest that homo- and heterodimerization occurs between cKrox family members. Dimer formation is influenced by amino acids in the NH(2)-terminal POZ domain and the Zn(+2)-finger region. Immunoprecipitation studies indicate that cKrox can form heterodimers in solution in the absence of DNA. Thus, a multi-gene family exists that can coordinately regulate several extracellular matrix genes and has the potential to form many heterodimeric transcription factors.