Following treatment of cultured mosquito cells (Aedes albopictus line of Singh) with the chemical mutagen ethyl methanesulfonate, we were able to isolate three cycloheximide-resistant clones. On the basis of growth kinetics, plating efficiency, and protein synthesis, these clones are 10- to 30-fold more resistant to cycloheximide than the parent cells. Cell-free lysates made from these cells retained 30-65% of their endogenous protein synthesizing ability in the presence of cycloheximide concentrations as high as 300 micrograms/ml. Protein synthesis in lysates from the parental cells, however, is reduced to about 10% of the control value (i.e., in the absence of drug) at 14 micrograms of cycloheximide/ml and was completely abolished at 75 micrograms/ml. These results indicate that cycloheximide resistance in these cells is likely due to an alteration in the protein synthetic machinery. This is the first description of cycloheximide-resistant insect cells, and the best example of cycloheximide resistance in cells originating from a higher eukaryotic organism.