The effect of interferon treatment on proteins synthesized in simian virus 40 (SV40)-infected cells in the presence of cytosine arabinoside was investigated. The following results were obtained: (i) In addition to previously described large tumor (T) antigen (94 kilodaltons) and small tumor (t) antigen (19 kilodaltons), a 62-kilodalton polypeptide was immunoprecipitated by SV40 anti-T antiserum from extracts of infected CV-1 and BSC-1 monkey kidney cells and transformed SV3T3 mouse cells. The 94-, 62-, and 19-kilodalton polypeptides were not precipitated with normal serum from extracts of infected cells, and they were not present in extracts of uninfected cells. (ii) The de novo synthesis of the 94-, 62-, and 19-kilodalton tumor antigens was inhibited in CV-1 and BSC-1 cells treated with interferon before infection; total cellular protein synthesis was not significantly affected by interferon treatment. The relative interferon sensitivity of the three polypeptides in lytically infected monkey cells was comparable; by contrast, interferon did not affect their synthesis in transformed mouse cells. (iii) The 62-kilodalton polypeptide was detected in monkey cells infected with the following strains of SV40: tsA30 at both 33 degrees C and 41 degrees C; wt 708, the parent of tsA30; dI 884; and wt 830, the parent of dI 884. The amount of the 62-kilodalton species relative to T antigen was significantly greater in tsA30-infected cells as compared to cells infected with other SV40 strains. (iv) T, t, and 62-kilodalton polypeptides were readily labeled with [(35)S]methionine during a 10-min pulse; in a subsequent chase, the (35)S-labeled 94-kilodalton T antigen was apparently converted to 89- and 84-kilodalton polypeptides but not to either the 62-kilodalton polypeptide species or t antigen. (v) Partial peptide maps suggest that the 62-kilodalton polypeptide and T antigen are closely related. (vi) In addition to the above described 62-kilodalton polypeptide, a 54-kilodalton polypeptide was also detected. However, the 54-kilodalton species appears to be of cellular origin because it was immunoprecipitated with both normal and anti-T antiserum from uninfected and lytically infected cells and from virally transformed cells.