CAD is a multifunctional protein which catalyzes the first three steps of de novo uridine biosynthesis. Rodent cells resistant to PALA, a specific inhibitor of the ATCase activity of CAD, overproduce the CAD protein and CAD mRNA as a direct result of the amplification of the CAD gene. In order to study the mechanism of CAD gene amplification, a functional Syrian hamster CAD gene was inserted into a cosmid vector using molecular cloning techniques. The cloned genes were assayed for biological function by fusing CAD-deficient Chinese hamster ovary (CHO) cell mutants with protoplasts of E. coli containing the CAD cosmids. Two clones with functional CAD genes were isolated and shown to contain inserts 40 and 45 kb long. The cloned genes could also be introduced into wild type CHO cells by selecting for cells which became resistant to high PALA concentrations in a single step. Transformations of mutant and wild type CHO cells contained multiple active copies of the donated Syrian hamster CAD genes in addition to their endogenous CHO CAD genes. The cloned genes in all transformants analyzed are integrated into host cell chromosomes at single locations defined by in situ hybridization. Independently isolated transformants contain the donated genes in different chromosomes. Co-transformation of CHO cells with two different genes by protoplast fusion is also shown to be possible.