Galactokinase activity is reduced in 12 independent clones of Chinese hamster ovary cells resistant to 2-deoxygalactose. The frequency of resistant colonies is increased with chemical mutagens. The resistant phenotype is stable in the absence of selection. There is an inverse correlation between the levels of galactokinase activity and the cloning efficiency in deoxygalactose. Cells with high resistance have 1% or less of the enzyme activity observed in the parental cells; while cells with low resistance have 10-30% galactokinase activity. Studies with tetraploid hybrid cells reveal that resistance to deoxygalactose is a recessive trait and that cells with high resistance do not complement those with low resistance. In cell lines with low resistance, the Km for galactose, Ki for deoxygalactose, Km for ATP, and thermolability were not significantly altered compared to sensitive parental cells. Although the possibility of mutation at the structural gene locus has not been ruled out, the reduced enzyme activity may also be due to mutation at a regulatory site which affects the number of galactokinase molecules per cell.