We have found that proline and the toxic proline analogue azetidine-2-carboxylate (AzC) are efficiently imported into Saccharomyces cerevisiae cells by four amino acid permeases, including two nitrogen-regulated permeases (PUT4 and GAP1) and two permeases that are regulated by the SPS sensor of extracellular amino acids (AGP1 and GNP1). In contrast to Agp1p, Gnp1p is not functionally expressed when cells are grown on media containing proline as sole nitrogen source. These findings have implications for the interpretation of studies using AzC to characterize nitrogen source-dependent regulation of amino acid uptake and of post-Golgi targeting and localization of amino acid permeases in yeast.