Intestinal inorganic phosphate (Pi) transport seems to be mainly mediated by the sodium/Pi-cotransporter NaPi2b. To verify this role, we have studied the combined effects of pH, phosphonoformate, and Pi deprivation on intestinal Pi transport. Rats were fed, ad libitum, three fodders containing 1.2, 0.6, or 0.1% Pi for 1, 5, or 10 days. Pi deprivation (0.1%) increased both sodium-activated and sodium-independent Pi transport in brush-border membrane vesicles from the duodenum and jejunum, for all three times. Alkaline pH (range 6.0-8.5) inhibited Pi transport, despite the increasing concentration of HPO₄= (NaPi2b substrate), while acidity increased transport when the concentration of PiT1/PiT2 substrate, H₂PO₄-, was at its highest. The effect of Pi deprivation was also maximal at acid pH, but both basal and upregulated transport were inhibited (70%) with phosphonoformate, an inhibitor of NaPi2b. PiT2 and NaPi2b protein abundance increased after 24 hours of Pi deprivation in the duodenum, jejunum, and ileum, while PiT1 required 5-10 days in the duodenum and jejunum. Therefore, while transporter expressions are partially correlated with Pi transport adaptation, the pH effect precludes NaPi2b, and PFA precludes PiT1 and PiT2 as the main transporters. Transport and transporter expression was also inconsistent when feeding was limited to 4 hours daily, because the 1.2% Pi diet paradoxically increased Pi transport in the duodenum and jejunum, but NaPi2b and PiT1 expressions only increased with the 0.1% diet. These findings suggest the presence of a major transporter that carries H₂PO₄- and is inhibited by phosphonoformate, and its expression correlates with Pi transport