T antigen, purified from KB cells infected with type 12 adenovirus, was characterized by chemical and physical methods. The immunologically active molecule had an ultraviolet-absorption spectrum characteristic of a protein, and was partially inactivated (i.e., approximately 85%) by trypsin (final concentration of 150 mug/ml). Centrifugation analyses indicated that the purified antigen was a single molecular species with an average sedimentation coefficient of 2.40S (measured by zonal centrifugation in sucrose gradients) and an S(20,w) of 2.58 determined by analytical centrifugation. The antigen was inactivated at temperatures above 40 C, it was stable between pH 5.7 and 8.8, and it had an isoelectric point of approximately 5.0, as determined by electrophoresis on cellulose polyacetate membranes.