dl-2-Hydroxy-(4-methylthio)butanoic acid (HMTBA) is a synthetic source of dietary methionine (Met) widely used in poultry nutrition. HMTBA is transported in the intestinal epithelium by the monocarboxylate transporter 1, after which its biological utilisation relies on its conversion to l-Met. This process involves stereospecific HMTBA oxidation to 2-keto-(4-methylthio)butanoic acid (KMB) and transamination to l-Met. In the present study, we examined HMTBA conversion to l-Met, further incorporation into cellular proteins and the regulation of both processes by HMTBA supplementation in differentiated intestinal Caco-2 cells. The results showed d- and l-HMTBA oxidation in the enterocytes, this process being up-regulated by HMTBA. The data also revealed that KMB transamination is not linked to a specific amino group donor. However, the branched-chain amino acid l-leucine is the preferred amino group donor. Furthermore, transamination was not affected by HMTBA availability. The incorporation of radioactivity from HMTBA into cellular proteins was not significantly different from that of l-Met and was not affected by HMTBA supplementation. In conclusion, the results reveal the capacity of Caco-2 cells to convert HMTBA to l-Met and the up-regulation of conversion by nutritional HMTBA supplementation, thus highlighting the contribution of the intestinal epithelium in the utilisation of HMTBA as a dietary source of Met.