DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 X 10(4) focus forming units per picomole. These results indicate that we have molecularly cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcinoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.