When lymphocytes obtained from W/Fu rats primed with BCG are cultured in the presence of PPD, they elaborate a factor that is capable of potentiating the specific in vitro generation of cytotoxic lymphocytes to syngeneic (C58NT)D lymphoma cells and to BN alloantigen. Purification of the factor, achieved by gel filtration on Sephadex G-100, was facilitated by using a serum-free culture condition and the removal of the specific stimulating antigen, PPD, after an initial incubation period. The factor isolated contains DNA by its absorption spectrum, resistance to trypsin and RNase, but complete susceptibility to DNAase, and by the presence of ethidium bromide-positive material in the purified sample. It displays a 260/280 nm absorption ratio of 1.6 and a m.w. estimate of 10,000 to 30,000. Electrophoresis of the purified factor on agarose gel yielded three ethidium bromide reactive bands. Data obtained following the slicing and elution of these bands, and then testing for potentiating activity indicated that two of the three bands contained potentiating activity.