Rat (3Y1) and hamster embryo brain cells were transformed by wild-type adenovirus type 12 or the DNA-minus temperature-sensitive mutant ts401. The ts401-transformed 3Y1 cells, but not the wild-type transformants, displayed a temperature-sensitive response with respect to the following characteristics of the transformed phenotype: morphology, saturation density, growth rate, cloning in soft agar, colony formation on plastic at low cell densities in 1% serum medium, and the T antigen(s). Temperature shift-down experiments showed that the density-dependent inhibition of growth of the ts401-transformed cells was reversible, as was, to some extent, the low efficiency of colony formation at low cell densities in 1% serum. Examination of hamster transformants for their ability to clone in soft agar at permissive and nonpermissive temperatures showed that this property was temperature dependent, again only in the ts401 transformants and not in the wild-type transformants. Alteration in uptake of 2-deoxyglucose or in intracellular cyclic AMP content was not a characteristic of the adenovirus-transformed phenotype in the 3Y1 cells. The findings suggest that an active 401 function is required for maintenance of the adenovirus-transformed cell pheno-type.