Pools of young (less than 60% replicated) and mature (60-90% replicated) replicating molecules of simian virus 40 (SV40) DNA have been treated at pH 12.2 in order to dissociate growing chains from the parental strands. The molecules are neutralized so that the parental strands can reassociate and they have then been isolated. They are covalently closed structures which sediment rapidly in alkaline sucrose gradients; however, the sedimentation rates are less than the sedimentation rate of SV40 DNA I. Isopycnic banding in CsCl-ethidium bromide and sedimentation velocity studies in the presence of various amounts of ethidium bromide indicate that these structures contain negative superhelical turns and several-fold-higher superhelix densities than SV40 DNA I (the covalently closed DNA molecule). These structures are those that would be predicted if nicking, unwinding, and sealing of the parental strands occurred as replication proceeded. These experiments provide a direct demonstration that there is a progressive decrease in the topological winding number which accompanies SV40 DNA replication.