SV40-transformed Chinese hamster C0631 cells pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) display SV40 DNA amplification. This study shows that following MNNG treatment elevated T antigen synthesis and a 4.5-fold reduction in the extent of its phosphorylation occurred in both pulse-labeled and steady-state-labeled cells. The decrease in phosphorylation was found to be inversely related to carcinogen concentration, i.e. an augmented carcinogen concentration brought about a gradual reduction in T antigen phosphorylation and elevated SV40 DNA amplification. Although the majority of phosphorylation sites on T antigen derived from carcinogen-treated cells were underexpressed, as demonstrated by two-dimensional phosphopeptide mapping, peptide 12 bearing phosphoThrl24, which is known to be essential for DNA replication, was overexpressed. Carcinogen-treated cells showed no changes in p53 synthesis, but it was phosphorylated to a lesser degree. Two-dimensional mapping revealed that the predicted N-terminal major phosphopeptide of p53 extracted from C0631 cells exhibited a lower chromatographic mobility than p53 phosphopeptides from SV40-infected monkey BSC-1 cells. In treated C0631 cells the Rf value of this phosphopeptide was higher than that of control p53. This finding could be ascribed to the failure to phosphorylate the corresponding amino acid residue in this peptide. Moreover, treatment did not affect the halflife of either T antigen or p53 proteins, but caused a dramatic rise in the expression of small t antigen, presumably due to amplification of SV40 DNA.