Influx of phenylalanine across the brush border of rabbit intestine is markedly reduced by treatment with 5 mM p-chloromercuriphenyl sulfonate (PCMBS). The effect is rapidly and completely reversed by dithiothreitol. Phenylalanine influx into PCMBS-treated tissue can be competitively inhibited by other neutral amino acids and follows saturation kinetics. PCMBS causes an increase in the apparent Michaelis constant from the value observed in control tissue but does not alter the maximal influx significantly. Treatment of the tissue with PCMBS leads to a significant reduction in the Na-sensitivity of the transport, and a number of results indicate that the major effect of the reagent is to cause a marked reduction in the affinity of the transport system for Na. The transport system can be partially protected against reaction with PCMBS by phenylalanine and tryptophan but not by methionine or norleucine. The results suggest that PCMBS reacts with a sulfhydryl group in the region of the transport site and may alter conformational changes associated with the binding of substrates.