To improve our knowledge of the simian virus 40 (SV40) late promoter control region, we took advantage of the fact that T antigen can be expressed with a heterologous promoter. We constructed three chimeric plasmids (pEMP-273, pEMP-LCAP-162, and pEMP-LCAP-113) each with a putative late promoter sequence positioned immediately upstream from the SV40 early gene coding region but in an orientation opposite to its natural orientation in the SV40 genome. After transfection of the recombinant DNA into HeLa or CV1 cells, T antigen accumulation, as scored by indirect immunofluorescence, was used as a functional test for promoter activity. We found that the sequence mapping from nucleotides 332 to 273 is not sufficient for promoting transcription of SV40 early gene but does potentiate the promoter activity of the 72-base-pair repeats in initiating the transcription at natural late cap sites. Considering that both plasmids pEMP-LCAP-162 and pEMP-LCAP-113 lack all of the sequence of the SV40 replication origin, we conclude that SV40 transcription can be mediated through a putative late promoter in the absence of the sequence for the SV40 replication origin.