NS3 (nonstructural protein 3) from the hepatitis C virus is a 3' --> 5' helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80% of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in K(D) = 2.7 +/- 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the K(D) for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (K(D) = 1.3 +/- 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (K(D) = 11.3 +/- 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding.