Nutritionists are constantly challenged to formulate diets that not only meet the nutritional requirement of animals but are also cost-effective and have a low environmental impact. To ensure adequate growth and health of fish, methionine (Met) supplementation using DL-methionine (DL-Met) or DL-methionine hydroxy analogue (DL-MHA) is a common practice, allowing for increased use of plant-based protein ingredients in aquafeed. Determining how much and how quickly these Met sources are transported in the fish intestine is a critical step in assessing their bioefficacy because the intestine is the most important location of nutrient absorption, and insufficient absorption in the gut would increase waste excretion. This study used radiolabeled flux assays and transporter gene expression to 1) characterize transport pathways of DL-Met and DL-MHA across ex-vivo intestinal segments, and 2) compare the transport kinetics between the two. Intestinal transport of DL-[14C]Met and DL-[14C]MHA were measured in the presence or absence of sodium at physiological apical/basal pH of 7.7/7.7, as well as various pH levels (6.0/6.0, 6.0/7.7 and 7.7/8.7) at the substrate concentration ranging from 0.2-20mM. The results demonstrated that DL-[14C]Met flux was primarily driven by Na+-dependent process, with flux rates the in the pyloric caeca (PC) and midgut (MG) regions being higher than in the hindgut (HG). On the other hand, the apical transport of DL-[14C]MHA appeared to be facilitated by Na+-requiring systems, whereas basolateral flux was governed by an H+-independent process in PC and MG, but an H+-dependent process in the HG. Finally, a comparison made under identical experimental conditions showed that the flux rates of DL-[14C]Met were significantly greater than that of DL-[14C]MHA in PC and MG, suggesting that the intestinal transport of DL-Met in trout is more efficient than its corresponding analogue (Table 1).