We isolated simian virus 40 (SV40) chromosomes from lytically infected CV-1 cells at various times during the late phase and transcribed them in vitro with either whole-cell or nuclear extracts of HeLa cells. The late promoter was 3- to 10-fold more active than the early promoter. With bare SV40 DNA templates, the early promoter was up to 10-fold stronger than the late promoter. The relative strengths of the early and late promoters on SV40 chromosomes were essentially independent of template concentration or length of the replicative phase of the infection. When monoclonal antibodies or antisera against T antigen (T Ag) were added to SV40 chromosomes or when T Ag, both free and chromatin bound, was removed by immunoprecipitation with anti-T, the activity of the late promoter remained essentially unchanged. Washing with 0.4 M NaCl removed T Ag from more than 90% of the mature chromosomes associated with T Ag. Transcription from the late promoter still predominated in the salt-washed T Ag-depleted chromosomes, even though there was a marked increase in early promoter activity. The depression of the early promoter could be reversed by adding the T Ag-containing extract back to the depleted chromosomes. Extraction of SV40 chromosomes with 1.5 M NaCl resulted in a decrease in the activity of the late promoter and a further increase in the activity of the early promoter so that the relative amounts of early and late RNA synthesized were similar to those for bare SV40 DNA templates. Late RNA synthesis from bare SV40 DNA templates was stimulated by high-speed supernatants prepared from nuclear extracts of SV40-infected cells but not from those of uninfected cells. Pretreatment of the supernatants with anti-T did not alter the result. Our findings indicate that the activity of the early and late SV40 promoters is regulated by at least two different mechanisms at the chromosomal level. One is mediated by a subclass of T Ag bound to SV40 chromosomes which represses early SV40 transcription but has no effect on late transcription. A second level of regulation, involving a tightly bound trans-acting chromosomal factor and a stable nucleoprotein structure, favors the late promoter over the early promoter by up to 10-fold.