Abstract 1. 1. The properties of a proline accumulating system in nitrogen starved cells of Saccharomyces chevalieri are described. The system shows a Michaelis-Menten substrate dependence with an apparent K m of 2.5·10 −5 M for l-proline and a K i of 8.9·10 −5 M for d-proline. 2. 2. The intracellular radioactivity accumulated corresponds to at least 85% free proline, as indicated by chromatography, displacement of accumulated proline and bio-assay. 3. 3. The uptake of proline is temperature dependent. Further, azide and 2.4-dinitrophenol inhibit the uptake. 4. 4. Measurement of the initial velocity of proline influx shows that the uptake of proline is not inhibited by a number of natural amino acids. Only α-alanine is inhibitory. l-Hydroxyproline and other amino acids are poor inhibitors. Some amino acids, such as tryptophan, phenylalanine and tyrosine, stimulate uptake. 5. 5. Among several structural analogs of proline, only sarcosine, l-thiazolidin-4-carboxylic acid, d- and l-azetidin carboxylic acid and 3,4-dehydro- dl-proline were found to be effective inhibitors of the system. All of these show a competitive type of inhibition. 6. 6. The results presented are consistent with the idea that the proline transport system in yeast is very specific.