We have defined a medium (called Sun's modified Waymouth medium) that selectively cultures some rodent epithelial cells that are capable of using citrulline in place of arginine. A growth-response study of the ability of 47 different mammalian cell cultures (of mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, monkey, and human origin) to use arginine or its biosynthetic precursors, ornithine, citrulline, or argininosuccinate, showed that all epithelial cells and some fibroblasts are capable of growing in citrulline medium; however, primary embryo fibroblasts and 12 established fibroblast cell lines derived from Syrian hamsters failed to grow. The citrulline medium also allowed selective outgrowth of epithelial cells, without contaminating fibroblasts, from Syrian hamster tracheal explants. This absolute nutritional difference between Syrian hamster epithelial and fibroblast cells allows citrulline medium to be used for selective cultivation of epithelial cells, which should be valuable for study of growth, differentiation, and malignant transformation of mammalian epithelial cells.