These studies examined cell cycle progression and quantitative changes in T-antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T-antigen expression. Conditions were used which permitted permissive, semi-permissive, and non-permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines; CV-1, Vero and BSC-1 and the COS-1 and COS-7 which are CV-1 cells transformed with an origin defective SV40. The non-permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR-90, a human diploid fibroblast. Cell types differed in the maximal amount of T-antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T-antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two-fold when 100 times more virus was used. Thus, for an infected population the quantity of T-antigen was dependent on cell cycle distribution. High levels of T-antigen were not required for permissive infection; however, permissive cells were distinguished from non-permissive cells by the G2 levels. Permissive G2 cells had more than double the T-antigen content expressed in G1, while nonpermissive G2 cells had less than a two-fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher T-antigen levels in the G2 of the permissive cells. Two other strains of SV40, 776, and VA45 exhibit similar values for T-antigen expression and movement into tetraploid DNA content. This study establishes the levels of T-antigen correlated to the cell cycle and cell type.