The second member of the PAT (proton-coupled amino acid transporter) family of H(+)-coupled, pH-dependent, Na(+)-independent amino acid transporters was isolated from a rat lung cDNA library. The cDNA for rat PAT2 is 2396bp in length, including 70bp of 5'UTR and a poly(A) tail. The transporter gene, consisting of 10 exons, is located on rat chromosome 10q22. The cDNA codes for a protein of 481 amino acids with 72% identity (over 449 amino acids) with rat PAT1. Tissue expression studies demonstrate that mRNA abundance is generally low with highest levels being detected in lung and spleen, with lower levels in the brain, heart, kidney, and skeletal muscle. Functional expression in either mammalian cells or Xenopus laevis oocytes demonstrates that rat PAT2 mediates pH-dependent, Na(+)-independent uptake of glycine, proline, and alpha(methyl)aminoisobutyric acid (MeAIB). In conclusion PAT2 has a limited tissue distribution, higher affinity (Michaelis-Menten constant for glycine uptake between 0.49 and 0.69mM), and distinct substrate specificity compared to PAT1.