Objectives In mammalian cells, aspartate transcarbamylase is part of a trienzymatic protein called CAD. A new method is described that allows for the selection of stably transfected cells expressing high levels of CAD.Design and Methods Hamster CAD cDNA was subcloned into a bicistronic expression vector along with the neomycin resistance gene. The resulting construct was transfected into a CAD deficient hamster cell line that requires uridine for growth. Medium containing G418 and lacking uridine was used for selection. An irreversible inhibitor of aspartate transcarbamylase, N-phosphonacetyl-L-aspartate, was also used to select for colonies overexpressing CAD.Results After transfection, colonies were observed that no longer required uridine for growth. Transfectants were isolated that were resistant to 1 mM inhibitor (wild-type cells have an LD 5 0 of 5 μM). Some of the colonies expressed levels of CAD 10 times higher than a normal hamster cell line.Conclusion An efficient, high expression system was developed which makes studying the structure and function of CAD easier.