Hamster tumor cells requiring glutamine for survival and growth were used in this study. By gradually reducing the glutamine concentration over a period of 3 weeks, it was possible to establish a line which grew in the absence of glutamine. These cells, in contast to the glutamine-dependent cells, showed parallel alignment which is typical of normal hamster embryo fibroblasts. They were, however, readily distinguishable from normal cells because they grew to higher maximum density, were more refractile when examined by phase-contrast microscopy, and were more readily dispersed with trypsin. Injection of these cells into hamsters resulted in the development of slow growing solid tumors. When cells from these tumors were explanted into tissue culture, they were found to have reverted to glutamine dependence and to display the random crisscross growth pattern which was typical of the tumor cell line from which they were originally derived.