WtSV40 and its variant EL-SV40 (contains two complementing defective genomes) fail to productively infect human embryonic kidney cells or human fibroblasts. However, early SV40 (E-SV40) genomes can propagate in human cells when complemented by a particular late RF virus (L-RFV) genome or the closely related wtBKV genome. The L-RFV genome (L-RFV clone H) contains a deleted early region, a complete set of BKV capsid genes, and a single SV40 regulatory region (acquired by recombination). In contrast, it was not possible to make the reciprocal genome cross in human cells; late SV40 genomes containing a deleted early region do not complement early RFV or early BKV DNAs. The L-RFV clone H genome was also shown to complement wtSV40 in human cells. However, wtSV40 DNA was rapidly lost and replaced by a defective SV40 genome. The SV40 defective (E-SV40α) contained a deletion of the late region, an intact early region, and paired with L-RFV clone H DNA to form a new hybrid virus. In human cells wtSV40 was also complemented by wtBKV DNA, but after two serial passages SV40 DNA disappeared. These findings indicate that SV40 late or capsid gene sequences, but not SV40 early sequences, generate a block to SV40 growth in human cells. When the SV40 late region is replaced by a RFV or a BKV late region, E-SV40 DNA propagates efficiently in human cells and in some cases more rapidly than wtBKV. Northern blot hybridization indicates that SV40 DNA is poorly transcribed in human cells when the SV40 late region is present