In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 X 10-9 mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Class I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait.