A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth. GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media. However, the GLN requirements of tumor cell lines and the factors that determine a cell's need for GLN have not been comprehensively studied. Also, it remains unclear how various metabolic pathways contribute to GLN consumption. In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin. These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates. GLN uptake was facilitated by a single, common transporter functionally defined as System ASC. However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines. Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity. The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor-derived cells. The rates of CO2 production from GLN were similar for each cell lines. Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32-80% of media GLN converted to glutamate. Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system x(c)-. These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis.