Lines of mouse L cells resistant to the fusion-inducing effect of polyethylene glycol (PEG) 1000 have been isolated by treating cultures with 50% PEG and selecting cells that remained unfused and able to proliferate. With repeated cycles of PEG treatment and selection, populations have been obtained that exhibit progressively increased resistance to the fusion-inducing effect of PEG. With the parental line, more than 90% of the cells fused when treated with 50% PEG. In contrast, with a population isolated after 24 cycles of PEG selection, fewer than 25% of the cells fused when treated with PEG. The decreased fusion response of the selected cells appeared to be stable for long periods of growth in the absence of PEG treatment, and the populations of selected cells seemed homogeneous in terms of the fusion response of individual subclones. Resistance to the fusion-inducing effect of PEG was concentration dependent, as the selected cells that fused poorly when treated with 50% PEG fused very efficiently when the PEG concentration was increased to 55%. The cells that were resistant to the fusion-inducing effect of 50% PEG also were more resistant to the toxic effect of PEG than were the parental cells.