We constructed a synthetic cruciform DNA which closely resembles Holliday junctions, a DNA structure formed during recombination or following the transition from interstrand to intrastrand base pairing in palindromic DNA sequences. We identified and partially purified a protein from rat liver that specifically binds to this cruciform DNA molecule and does not bind to single-stranded or double-stranded DNAs of the same sequence. This protein also binds to the cruciform structure formed by a 70 bp palindromic sequence cloned in plasmid pUC18. No detectable nucleolytic activity is associated with the rat liver cruciform-binding protein, in contrast to all cruciform-recognizing proteins known so far.