The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.