The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports L-serine, L-threonine, and L-cysteine with high affinity. Affinity constants (K m ) are in the 20 to 40 M range. SerP2 is a DLalanine/DL-serine/glycine transporter. The preferred substrate appears to be DL-alanine for which the affinities were found to be 38 and 20 M for the D and L isomers, respectively. The common substrate L-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 M, respectively. Growth experiments demonstrate that SerP1 is the main L-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates L-alanine and glycine. T he lactic acid bacterium (LAB) Lactococcus lactis is extensively used for the manufacture of buttermilk and cheese. L. lactis originally lived on plants, and dairy strains are currently derived by reductive evolution (1, 2). The change to nutrient-rich environments like milk is believed to be the reason that dairy strains of L. lactis lost biosynthetic pathways for various amino acids like branched-chain amino acids and histidine (3, 4). In milk, casein provides a rich source of all amino acids. It is degraded by an efficient proteolytic system, which consists of a proteinase, several peptidases, and a peptide transport system. The proteinase PrtP is attached extracellularly to the cell wall and hydrolyzes casein to peptides varying in length from 5 to more than 30 amino acid residues. The peptides are transported into the cell via the oligopeptide uptake system Opp and further degraded in the cell by intracellular peptidases (5, 6). The system generates free amino acids in the cytoplasm following uptake of peptides without the need for specific transporters to take up free amino acids from the environment. Nevertheless, media containing free amino acids as the sole source of amino acids do support growth of L. lactis (7), indicating that transport systems for single amino acids have been preserved. The physiological roles of many of these transporters during growth on different media are not clear.Transport studies in L. lactis and other LAB, mainly performed in the late 1980s, using whole cells or membrane vesicles demonstrated proton gradient-driven uptake activity for branched-chain amino acids and L-methionine (8, 9), L-alanine and glycine (10), L-lysine (11), L-serine, L-threonine, L-histidine, and L-cysteine (12), and L-tyrosine and L-phenylalanine (13). L-Glutamate, Lglutamine (14), and possibly L-proline (15) transport was shown to be driven by ATP hydrolysis. More recent work has identified many of the genes encoding the transporters. bcaP and gnlPQ were identified in L. lactis as encoding a secondary branche...