We have partially purified a DNA helicase BstH1 from Bacillus stearothermophilus through Polymin P precipitation, ammonia sulfate precipitation and column chromatographic steps with Pheny1-Sepharose, DEAE-cellulose, phosphocellulose, FPLC Mono Q and Superose 12. Bsth1 possesses a DNA-Dependent ATPase activity in the presence of Mg(2+). The ATPase activity of BstH1 is differentially stimulated by the presence of different types of nucleic acids. BstH1 has an optimal ATPase activity at 55 degrees. The DNA helicase activity of BstH1 requires a 3'-terminal single-stranded DNA binding site to initiate the unwinding reaction in the 3' right curved arrow 5'direction. BstH1 can unwind blunt-ended duplex DNA in a concentration-dependent manner.