Background & Aims: Intestinal glycine transport is involved in nutrient absorption and enterocyte homeostasis, particularly for glutathione synthesis. The primary aim of this study was to characterize the mechanism of postabsorptive (basolateral) glycine acquisition by the enterocyte. Methods: Assimilation of [14C]glycine was studied in human enterocytic Caco-2 cells, and expression of the glycine transporter GLYT1 was examined in Caco-2 cells and human intestine by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. The regulation of glycine transport in Caco-2 cells by phorbol-ester-induced protein kinase C activation was investigated. Results: Basolateral glycine uptake into Caco-2 cells is predominantly Na+ and Cl- dependent and is 4-fold greater than apical uptake. The dominant Na+- and Cl--dependent mechanism was characterized by a restricted inhibition profile, selectively sensitive to sarcosine, with an apparent Michaelis constant of 40-80 μmol/L, indicating system GLY. Consistent with these functional data, molecular techniques detected expression of GLYT1 messenger RNA and protein in the human intestine and Caco-2 cells. Protein kinase C activation reduced maximum velocity for GLYT1-mediated glycine uptake without effect on the Michaelis constant. The reduction in functional activity was independent of a measured protein kinase C-induced decrease in GLYT1 messenger RNA levels. Conclusions: Enterocytes express GLYT1 along the length of the crypt-villus axis, where it mediates high-affinity basolateral glycine uptake.