Intestinal tissue of normal (vitamin D-replete) chicks was analyzed for responsiveness to PTH by the criteria of phosphate transport in perfused duodenal loops and binding of iodinated hormone to isolated basal lateral membranes. Using the N-terminal fragment of the bovine hormone [bPTH-(1-34)], the peptide agonist was found to stimulate phosphate transport within 4 min of perfusion (P < 0.05), reaching 152% of the control level after 40 min of exposure to 65 pM hormone. Dose-response analyses revealed an apparent optimum between 65-650 pM bPTH-(1-34), with decreased responsiveness at 6.5 nM hormone. Perfusion of duodenal loops with 65 pM 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] also stimulated phosphate transport, although a combination of both hormones did not have additive effects. Augmented phosphate transport mediated by bPTH-(1-34) was not attenuated by simultaneous perfusion with 6.5 nM 24,25-(OH)2D3, in contrast to earlier findings with 1,25-(OH)2D3-stimulated phosphate transport. Subcellular fractionation of duodena vascularly perfused with control medium or 65 pM bPTH-(1-34) for 5 min revealed hormone-augmented levels of 32P in lysosomal and mitochondrial fractions and decreased soluble 32P relative to control levels. In parallel studies, 65 pM 1,25-(OH)2D3 resulted in qualitatively similar subcellular distributions of radionuclide, whereas simultaneous perfusion with 65 pM 1,25-(OH)2D3 and 6.5 nM 24,25-(OH)2D3 abolished the increase in lysosomal 32P, but not augmented mitochondrial, levels. These findings suggest that lysosomes are vesicular phosphate carriers. Finally, preparation of isolated basal lateral membranes for binding studies with 125I-labeled [Nle8.18,Tyr34]bPTH-(1-34) amide indicated specific saturable binding with a Kd of 0.2 nM. At 1000-, 100-, and 10-fold excesses of unlabeled competitor, bPTH-(1-34) was 90%, 80%, and 33%, respectively, as effective as the analog in displacing labeled peptide. The combined data suggest that PTH may play a significant physiological role in regulating intestinal phosphate absorption.