The enzyme(s) RNA‐dependent DNA polymerase (reverse transcriptase) has been isolated from human lymphocytic cell lines which have been derived from patients with leukemia, infectious mononucleosis, and from normal healthy donors. The reverse transcriptase was purified through high‐speed glycerol gradients, and by DEAE‐cellulose and phosphocellulose column chromatography. The enzyme isolated from the cultured human lymphocytes had identical chromatographic patterns. The enzyme properties were studied by utilizing synthetic templates. These synthetic templates are known to discriminate between reverse transcriptase activity and that from other cellular polymerases. The activity of reverse transcriptase from human leukemic cells was much greater than that of the enzyme isolated from cells of normal or infectious mononucleosis origin. The reverse transcriptase isolated from human leukemic cells preferred oligo(dT) · poly(dA) over templates oligo(dT) · poly(dA) and oligo(dG) · poly(rC), wherease templates oligo(dT) · poly(dA) and oligo(dG) · poly(rC) were found to be active with the enzyme isolated from normal cells. The detection of the enzyme(s) reverse transcriptase in well‐established cell lines of normal and malignant origin and its possible relation to cellular metabolism is discussed.