l-Proline is transported into the yeast Saccharomyces cerevisiae against a concentration gradient of up to 135:1, the gradient decreasing with increasing proline concentration and suspension density. The concentrative uptake is practically unaffected by inhibitors, except antimycin. It is markedly reduced by anaerobic conditions. Uptake of l-proline, either by normal cells or in the presence of inhibitors, elicits no alkalification of the medium (estimated by pH and conductivity measurements) and no membrane depolarization (estimated by distribution of tetraphenylphosphonium). There is no relationship between the electrochemical potential gradient of protons and the measured accumulation ratios of proline. Likewise, intracellular ATP levels bear little relation to the accumulation. If, based on analogy with other yeasts and bacteria, l-proline is symported with H + ions the process must occur in local domains of the membrane where both the ΔpH and the membrane potential may differ substantially from those measured in the bulk solution.