PcrA is an essential helicase in Gram-positive bacteria, but its precise role in cellular DNA metabolism is currently unknown. The Staphylococcus aureus PcrA helicase has both 5′→3′ and 3′→5′ helicase activities. In this work, we have studied the binding of S.aureus PcrA to a variety of DNA substrates that represent intermediates in DNA replication, repair, recombination and transcription. PcrA bound poorly or not at all to single-stranded DNA, double-stranded DNA with blunt ends, partially double-stranded DNA containing fork and bubble structures, and duplex DNA substrates containing either 5′ or 3′ single-stranded oligo dT tails. Interestingly, PcrA bound with high affinity to partially duplex DNA containing hairpin structures adjacent to a 6 nt long 5′ single-stranded region and one unpaired nucleotide (flap) at the 3′ end. However, PcrA did not detectably bind to partial duplexes with folded regions adjacent to a 6 nt long 3′ single-stranded tail (with or without a 1 nt flap at the 5′ end). PcrA showed moderate helicase activity with partially double-stranded DNAs containing 3′ or 5′ single-stranded oligo dT tails, the 3′→5′ helicase activity being more efficient than its 5′→3′ helicase activity. Interestingly, PcrA showed maximal helicase activity with substrates containing folded structures and 5′ single-stranded tails, suggesting that its 5′→3′ helicase activity is greatly stimulated in the presence of specific structures. However, the 3′→5′ helicase activity of PcrA did not appear to be affected by the presence of folded substrates containing 3′ single-stranded tails. Our data indicate that PcrA may recognize DNA substrates with specific structures in vivo and its 5′→3′ and 3′→5′ helicase activities may be involved in distinct cellular processes.