We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.