Hypervariable minisatellite DNA sequences are short, tandemly repeated sequences present at numerous loci in eukaryotes. They stimulate intermolecular homologous recombination up to 13-fold in human cells in culture and may be specific sites for the initiation of recombination in the eukaryotic genome (Wahls, W.P., Wallace, L.J., & Moore, P.D. (1990) Cell 60, 95-103). Reported here is the detection and partial purification of two hypervariable minisatellite DNA binding proteins, called Msbp-2 and Msbp-3, present in the nuclear extracts of human HeLa cells. The proteins elute from a gel filtration column with a native mass of 200-250 kDa and have sizes of 77 kDa and 115 kDa respectively.