Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F(-)) HIV-1(LAI) was completely defective for viral spread in the MT-4 T-cell line, yet F(-) HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1(NL4-3) and a derivative of HIV-1(HXBc2) deficient for both Vpr and Nef. In stark contrast to the previous report, F(-) derivatives of both strains replicated efficiently in MT-4 cells. F(-) HIV-1(NL4-3) also spread like wild-type HIV-1(NL4-3) in infected Jurkat and primary T-cell cultures. In contrast, F(-) HIV-1(HXBc2) was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F(-) HIV-1(HXBc2) entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F(-) HIV-1(HXBc2) growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type nef restored replication to F(-) HIV-1(HXBc2) in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.