A high mobility group (HMG) nonhistone protein fraction HMG(1 + 2), composed of HMG1 and HMG2, was prepared from pig thymus chromatin. In order to examine a possibility that the HMG(1 + 2) participates in the unwinding of the DNA double-helix, DNA hydrolysis assay systems with the endonucleases specific for single-stranded DNA were employed. In the presence of HMG(1 + 2), the hydrolysis of double-stranded DNA by N. crassa endonuclease was markedly promoted, while the hydrolysis of single-stranded DNA was hardly enhanced. The reaction kinetic data showed that the stimulation of the hydrolysis of double-stranded DNA in the presence of HMG(1 + 2) was due to the unwinding of the DNA double-helix by the HMG(1 + 2), and not due to stimulation of enzyme activity of the endonuclease by the protein. The unwinding reactions were dependent on the HMG protein concentration at low weight protein to DNA ratios and reached a maximum at the ratio of 0.025. The region unwound in the whole DNA was partial. Similar results were obtained for experiments with nuclease S1. Isolated HMG1 and HMG2 fractions showed DNA unwinding activity of similar extents. The association constant obtained by fluorescence quenching analysis showed that the HMG(1 + 2) has higher affinity to single-stranded DNA than to double-stranded DNA. The susceptibility to the unwinding differed with the DNA source. These results suggest that HMG(1 + 2) at a low weight protein to DNA ratio binds to some limited double-stranded region in DNA and unwinds the DNA partially.