The insect growth regulator diflubenzuron was found to be a potent inhibitor of melanosome synthesis and release in mouse melanoma cell cultures, and after three to five successive passages of melanoma cells in growth medium containing this compound, these cells were unable to produce monolayers in untreated medium and were incapable of inducing tumor formation in mice. This is the first time that an insect growth regulator has been shown to have a deleterious effect on malignant cells of animals.Because the insect growth regulator diflubenzuron (Dimilin; TH 6040; 1-(4-chlorophenyl)-3-(2,6-diflubenzoyl)-urea) has been found to disrupt insect epidermal cell histogenesis (4), we suspected that this compound also has a deleterious effect on melanoma cells, which, like the epidermal cells of insects, are of ectodermal origin (6). Thus, two murine melanoma cell cultures were exposed through several cell passages to diflubenzuron (DFB). These two cultures were the melanotic B16-F1 and the amelanotic B16-A melanoma cells.The melanoma cell cultures (low, <8 passages) were grown in Dulbecco modified Eagle medium (high glucose) supplemented with 10% fetal calf serum, 100 U of penicillin per ml, and 100 ,ug of streptomycin (GIBCO Laboratories, Grand Island, N.Y.) per ml. Cultures were harvested with the aid of a trypsin-EDTA solution (0.05 and 0.02%, respectively), suspended in Dulbecco medium, and seeded at concentrations of 105 cells per 100-mm polystyrene culture dish (Corning Glass Works, Corning, N.Y.), and incubated at 37°C in an atmosphere of 5% CO2 and 95% air. After 24 h of incubation, the medium was decanted, and the cells were divided into three groups. One group received Dulbecco growth medium, the second group received medium containing 0.1% dimethyl sulfoxide (Aldrich Chemical Co., Milwaukee, Wis.), and the third group received medium containing 12.5 ,ug of DFB (Thompson-Hayward Chemical Co., Kansas City, Kans.) per ml dissolved in dimethyl sulfoxide. After an additional 48 h of incubation, the medium was removed, and the cells were harvested as described above.Cells from each treatment were enumerated with the aid of a hemacytometer, and viability was determined by trypan blue exclusion and plating efficiency tests. Protein concentrations were determined by the Bio-Rad system (1), and melanin content was determined by spectrophotometric analysis (3). Spectrophotometric measurements were also made on incubation medium to determine the amount of melanin secreted. The control medium for determination of melanin content of the growth medium was the medium in which the B16-A amelanotic cells had been grown. Cells were harvested from three plates of each treatment for each determination. Six C57BL mice were inoculated subcutaneously with 0.2-ml cell suspensions (104 viable cells) from each group. Cells from each group were then replated in the same medium from which they were harvested, at concentrations of 105 cells per plate. This procedure was repeated until DFB caused some measurable effect on the cell culture...