The mechanism of glycine transport in lactating mouse mammary gland was investigated. Three Na+-dependent systems of glycine transport, distinguished on the basis of their ionic requirement and sensitivity to 2-(methylamino)isobutyric acid (MeAIB), were A (Na+-dependent, MeAIB-sensitive); (Na++Cl−)-dependent, MeAIB-insensitive; and Na+-dependent, Cl−-independent, MeAIB-insensitive. These systems were further distinguished on the basis of inhibition analysis and sensitivity to pH of the extracellular medium and preloading mammary tissue with amino acids. The uptake of glycine via the A system (Km 0·53 mM) was inhibited by preloading mammary tissue with alanine, while glycine uptake mediated by the (Na++Cl−)-dependent, MeAIB-insensitive system (Km 0·47 mM) was downregulated by preloading mammary tissue with all amino acids (alanine, sarcosine and histidine) tested. Treatment of mammary tissue with N-ethylmaleimide inhibited the uptake of glycine via both these systems. Decreasing the pH of the extracellular medium inhibited the uptake of glycine via the A system but not the (Na++Cl−)-dependent, MeAIB-insensitive system. On the basis of ionic requirement, system A appears to comprise two components, one dependent on Na+ plus Cl− and the other on Na+ alone. Insulin upregulated the A system-mediated uptake of glycine in pregnant mouse mammary tissue cultured in vitro, while the (Na++Cl−)-dependent, MeAIB-insensitive system remained unaffected.