Within the flower, microsporogenesis represents a major sink for nitrogen, but knowledge on how the imported nitrogen is transferred from the anther cell layers to developing pollen is lacking. Here, we provide information on characterization of a transporter (AtLHT2) that might play an important role in partitioning of amino acids for microspore development. Biochemical analysis in yeast showed that AtLHT2 transports proline and aspartate with high affinity. However, other neutral and acidic amino acids act as strong competitors for proline and aspartate uptake indicating that AtLHT2 generally transports uncharged and negatively charged amino acids. Comparison of the apparent K(m) values of AtLHT2 with previously characterized amino acid transporters clearly demonstrated that AtLHT2 represents a novel high-affinity system for neutral and acidic amino acids. Northern blot analysis showed strong expression of the amino acid transporter in flower buds. Cellular expression could be resolved by using RNA in situ hybridization and in situ RT-PCR methods, which localized AtLHT2 specifically to the tapetum tissue of the anthers. Developing pollen grains are symplasmically isolated from the sporophytic tissue and rely on the nutrients and other compounds secreted from the tapetum cells. Thus, the functional characterization of AtLHT2, together with our expression and localization studies, strongly suggest that in Arabidopsis flowers, AtLHT2 has a critical function in import of neutral and acidic amino acids into the tapetum cells for synthesis of compounds important for microspore structure and in transfer of organic nitrogen to the locule for pollen development.