We have characterized the transcellular transport of a large neutral amino acid (LNAA) in Caco-2 cell monolayers. The apical (AP) to basolateral (BL) transport of phenylalanine (Phe) was approximately 10-times faster than BL-to-AP transport. The evidence for the carrier-mediated AP-to-BL transport of Phe include: (a) temperature dependence and saturability, (b) Phe transport was not affected by a reverse gradient, (c) the activation energy for transport was 12.0 kcal/mol, and (d) an excess amount of unlabeled Phe caused a 75% reduction in transport rate and a delay (lag time) in the appearance of Phe in the BL side. The Vm and Km for Phe transport were 572.4 pmol.mg protein-1.min-1 and 0.56 mM, respectively. Phe transport was decreased in the absence of glucose and in the presence of sodium azide or ouabain. The carrier interacted with LNAAs and with cationic amino acids but not with small neutral or anionic amino acids.