Overexpressed PIF1 DNA helicase was purified from mitochondria to near homogeneity. Its ATPase and unwinding properties were characterized. The enzyme specifically utilizes ATP (or dATP) and MgCl2 (and to a lesser extent MnCl2). ATPase activity requires single-stranded DNA as an effector, duplex DNA being 100-fold less effective. The Keff, defined as the concentration of DNA required to achieve half-maximal ATPase activity, does not depend on single-stranded DNA length. Long duplex DNAs are poorly unwound and, moreover, dilution of the enzyme and its DNA substrate in the assay decreases DNA helicase activity. These data indicate that PIF1 helicase is a distributive enzyme, frequently turning from one DNA molecule to another. When forked substrates are used, unwinding by PIF1 is markedly stimulated. The enzyme has a sedimentation coefficient of 6.5 S, suggesting that it exists as a monomer in solution.