A DNA helicase, called DNA helicase alpha, was purified from HeLa cells to apparent homogeneity. The helicase and its single-stranded DNA-dependent ATPase activities cosedimented in glycerol gradients with two polypeptides of 110 and 90 kDa with a sedimentation coefficient of 7.4 S. The DNA helicase was markedly stimulated by DNA substrates with a 5'-tailed fork. A DNA substrate with a 3'-tailed fork structure was less stimulatory, although it was more active than substrates without a fork. The directionality of unwinding is 3' --> 5' with respect to the single-stranded DNA to which the enzyme was bound. The helicase activity also required a single-stranded DNA-binding protein (SSB) for unwinding activity. The stimulation by SSBs was nonspecific; all SSBs tested, such as human SSB, bacteriophage T4 gene 32, and Escherichia coli SSB, stimulated the DNA helicase activity to a varying extent in the presence of a fork structure. With long duplex substrates (> 500 base pairs), the presence of a fork substantially stimulated the DNA helicase activity in the presence of E. coli SSB. Human SSB stimulated the DNA helicase activity to the greatest extent (> 10-fold) with a substrate containing a fork compared with substrates without a fork. DNA helicase activity required ATP hydrolysis and could be supported by all eight nucleoside triphosphates. The K(m) values for ATP and dATP in unwinding were 28 and 48 muM, respectively. In general, ribonucleoside triphosphates were better effectors than deoxyribonucleoside triphosphates. The properties of this DNA helicase make it a candidate for a DNA replicative helicase in human cells.