The expression of p53 in human cells infected with wild-type (wt) and mutant adenoviruses has been examined. With wt Ad5 and Ad12, and Ad12 viruses carrying lesions in the E1A or the 19K E1B genes, there was a pronounced decrease in level of p53 during the course of infection. However, when cells were infected with mutant viruses which did not express the larger E1B proteins (Ad12 dl620 and in602 and Ad5 dl338 and pm381) the concentration of p53 increased markedly to levels comparable to those seen in adenovirus transformed cells. This increase in level of p53 correlated closely with the advent of E1A expression. Infection with Ad5 dl355 (which carries a lesion in the E4 gene) also resulted in an increase in p53 expression. We have concluded that these results can be explained on the basis of the known ability of E1A to stabilize p53 and of the E1B 58K:E4 34K protein complex to regulate mRNA metabolism during viral infection, although large increases in expression of p53 or any other cellular proteins following infection with these viruses have not previously been reported. It is suggested that the high concentrations of p53 could explain the inability of 54K and 58K negative mutants to transform cells in culture. In cells infected with dl355 both the Ad5 E1B 58K protein and p53 were located in the nucleus. It was shown by coimmunoprecipitation experiments that these proteins formed a complex which was stable in the presence of high concentrations of NaCl. The interaction of the Ad12 E1B 54K protein and p53 has also been demonstrated in Ad12 E1-transformed cells by immunoprecipitation experiments. These data, taken in conjunction with previous results, have suggested that increased expression of p53 is unrelated to complex formation with the larger Ad E1B proteins.