We used 21 monoclonal antibodies (PAbs 100 to 117, 405, 419, and KT3) specific for different determinants in simian virus 40 (SV40) large T antigen (T) and one antibody specific for p53 that coprecipitates T complexed with p53 (T-p53) to analyze T in SV40-infected CV1 cells. We measured the ATPase specific activity, extent of adenylylation, and p53 content of T precipitated by antibodies directed against the N-terminal region I (0.65 to 0.62 map units), the midregion III (0.43 to 0.28 map units) containing both the ATPase- and nucleotide-binding sites, and the C-terminal region IV (0.28 to 0.17 map units) of T. Lytic T appeared to exist in three different forms with respect to p53 binding and ATPase activity. The most ATPase-active form of T was that precipitated by PAb 122. This T-p53 complex contained only 6% of the total T but contributed 35% of the ATPase activity, on average. Free p53 isolated from 3T6, Ann-1, or L929 cells had no apparent ATPase activity. A second form of T precipitated by several antibodies had little associated p53 but appreciable ATPase activity, accounting for 15 to 20% of total T and 60 to 70% of the ATPase activity. The rest of T constituted the third form and was also depleted in p53 but had a decreased ATPase specific activity. Thus, the remaining 75 to 80% of T had 15 to 20% of the ATPase specific activity. Antibodies specific for region III precipitated T with both altered ATPase activity and altered amounts of bound p53. PAbs 104 and 114 reacted with ATPase-active T but inhibited ADP hydrolysis, suggesting that they were inactivating antibodies. T that was preferentially adenylylated in vitro corresponded to T that was also preferentially ATPase active. T bound to p53 was adenylylated to a higher specific activity than total T. In addition, p53 itself was significantly adenylylated under these conditions. The results suggest that ATPase activity and p53 binding are structurally and functionally related and that p53 alters biochemical activities of T and plays a role in productive infection.