Supernatants of established cultures of human neoplastic and normal cells have been shown to contain a number of different biological activities, including inhibition of DNA synthesis as measured by thymidine uptake. We have found that supernatants of melanoma cell lines contain an inhibitor of thymidine uptake which is heat labile, ultraviolet sensitive, non-filtrable (0.22 mu) and partially sedimentable at 20,000 x g. The mechanism of action of the inhibitor involves the degradation of 3H-thymidine to a non-utilisable form, which we detect by failure of uptake of 3H-thymidine by cultures of mitogen stimulated lymphocytes to which the inhibitor is added. While microbiological tests have failed to reveal mycoplasma contamination of inhibitor producing cultures, treatment of these cultures with kanamycin suppresses inhibitor production. Qualitative evaluation of DNA synthesis by the inhibitor producing cultures using autoradiography and fluorescent DNA staining has confirmed the presence of mycoplasma. With the widespread use of established cell lines in cancer research, it is imperative that screening for mycoplasma contamination go beyond routine microbiological assays. Detection of 3H-thymidine degradation by cell culture supernatants is an additional simple and sensitive indirect test which could be used for this purpose.