The simian virus 40 (SV40) DNA segments present in a series of adenovirus-SV40 hybrids have been mapped with respect to the sites of cleavage of SV40 DNA by restriction endonucleases. Two approaches have been used. First, nucleic acid hybridizations were performed between equimolar quantities of the denatured DNAs of SV40 and each hybrid virus and the radiolabeled transcripts of 11 DNA fragments obtained by cleavage of SV40 DNA by restriction endonuclease from Hemophilus influenzae. Secondly, selected fragments of SV40 DNA produced by the H. influenzae or H. parainfluenzae restriction endonucleases were used to form heteroduplex DNA molecules with adenovirus and adenovirus-SV40 hybrid DNA, which were then analyzed by electron microscopy. The two sets of data were consistent and have permitted alignment of the map of the SV40 segments of the hybrid viruses with the H. influenzae and H. parainfluenzae cleavage maps of SV40. Since cells infected with some of the hybrid viruses contain one or more SV40-specific antigens, the genetic determinants of these antigens could be localized on the cleavage map.