The transport of L-glutamine by the lactating rat mammary gland has been investigated using rat mammary tissue explants and the in situ perfused rat mammary gland. L-glutamine uptake by both explants and the perfused mammary gland was via both Na+-dependent and Na+-independent pathways. It appeared that these pathways are situated on the blood-facing aspect of the mammary gland. L-glutamine uptake by both mammary preparations was markedly inhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid in the absence of external Na+. This is consistent with L-glutamine uptake via system L. The Na+-dependent component(s) of L-glutamine uptake remains to be precisely identified. However, system A can be ruled out on the basis that L-glutamine was not inhibited by (methylamino)isobutyric acid. Mammary tissue concentrates L-glutamine with respect to both milk and plasma: we suggest that the Na+-dependent component(s) of L-glutamine uptake is responsible for generating the intracellular to extracellular concentration gradient.