Aquaporin (AQP)-mediated intestinal water absorption may play a major osmoregulatory role in euryhaline teleosts, although the molecular identity and anatomical distribution of AQPs in the fish gastrointestinal tract is poorly known. Here, we have investigated the functional properties and cellular localization in the intestine of two gilthead seabream (Sparus aurata) homologs of mammalian aquaporin-1 (AQP1), named SaAqp1a and SaAqp1b. Heterologous expression in Xenopus laevis oocytes showed that SaAqp1a and SaAqp1b were water-selective channels. Real-time quantitative RT-PCR and Western blot using specific antisera indicated that abundance of SaAqp1a mRNA and protein was higher in duodenum and hindgut than in the rectum, while abundance of SaAqp1b was higher in rectum. In duodenum and hindgut, SaAqp1a localized at the apical brush border and lateral membrane of columnar enterocytes, whereas SaAqp1b was detected occasionally and at very low levels at the apical membrane. In the rectum, however, SaAqp1a was mainly accumulated in the cytoplasm of a subpopulation of enterocytes spreaded in groups over the surface of the epithelia including the intervillus pockets, while SaAqp1b was detected exclusively at the apical brush border of all rectal enterocytes. Freshwater-acclimation reduced the synthesis of SaAqp1a protein in all intestinal segments, but it only reduced SaAqp1b abundance in the rectum. These results show for the first time in teleosts a differential distribution and regulation of two functional AQP1 homologs in the intestinal epithelium, which suggest that they may play specialized functions during water movement across the intestine.