Nuclear import of plasmid DNA in nondividing cells is a process essential to the success of numerous viral life cycles, gene therapy protocols, and gene expression experiments. Here, intact protein-free SV40 DNA was cytoplasmically injected into cells and its subcellular localization was followed by in situ hybridization. SV40 DNA localized to the nucleus consistent with a mechanism of transport through the nuclear pore complex (NPC): import was inhibited by the addition of the NPC-inhibitory agents wheat germ agglutinin and an anti-nucleoporin antibody as well as by energy depletion. DNA transport appeared to be a multistep process with the DNA accumulating at the nuclear periphery before its import. Most importantly, nuclear import was sequence specific: a region of SV40 DNA containing the origin of replication and the early and late promoters supported import, whereas bacterial sequences alone and other SV40-derived sequences did not. The majority of the imported DNA colocalized with the SC-35 splicing complex antigen, suggesting that the intranuclear DNA localizes to areas of transcription or message processing. This link to transcription was strengthened by the finding that inhibition of transcription blocked DNA import but not protein nuclear import. Taken together, these results support a model in which plasmid DNA nuclear import occurs by a mechanism similar to that used by nuclear localization signal-containing proteins but is also dependent on transcription.