Despite the importance of extracellular phosphate in many essential biological processes, the mechanisms of phosphate transport across the epithelium of different intestinal segments remain unclear. We have used an in vitro method to investigate phosphate transport at the brush border membrane (BBM) of intact intestinal segments and an in vivo method to study transepithelial phosphate absorption. We have used micromolar phosphate concentrations known to favor NaPi-IIb-mediated transport, and millimolar concentrations that are representative of the levels we have measured in luminal contents, to compare the extent of Na(+)-dependent and Na(+)-independent phosphate transport along the rat duodenum, jejunum, ileum, and proximal and distal colon. Our findings confirm that overall the jejunum is the main site of phosphate absorption; however, at millimolar concentrations, absorption shows ~30% Na(+)-dependency, suggesting that transport is unlikely to be mediated exclusively by the Na(+)-dependent NaPi-IIb co-transporter. In the ileum, studies in vitro confirmed that relatively low levels of phosphate transport occur at the BBM of this segment, although significant Na(+)-dependent transport was detected using millimolar levels of phosphate in vivo. Since NaPi-IIb protein is not detectable at the rat ileal BBM, our data suggest the presence of an as yet unidentified Na(+)-dependent uptake pathway in this intestinal segment in vivo. In addition, we have confirmed that the colon has a significant capacity for phosphate absorption. Overall, this study highlights the complexities of intestinal phosphate absorption that can be revealed using different phosphate concentrations and experimental techniques.