The expression of P-gp increases from proximal to distal parts of the small intestine, whereas for P450 enzymes the expression is reported to be highest in duodenum and jejunum, decreasing to more distal sites. To evaluate to what extent the regional differences in expression of P-gp and P450 enzymes affect the absorption of a dual substrate, we investigated the transport of darunavir across different small intestinal segments (duodenum, proximal jejunum and ileum). Moreover, the effect of ketoconazole on the intestinal absorption of darunavir was explored, since these drugs are commonly co-administered. Performing the rat in situ intestinal perfusion technique with mesenteric blood sampling, we found no significant differences in the transport of darunavir at the different intestinal segments. The involvement of P-gp in the absorption of darunavir was clearly shown by coperfusion of darunavir with the P-gp inhibitor zosuquidar. In presence of zosuquidar, a 2.2-, 4.2- and 5.7-fold increase in Papp values were measured for duodenum, proximal jejunum and ileum, respectively. Involvement of P450 mediated metabolism in the absorption of darunavir could not be demonstrated in this rat model. Upon studying the drug–drug interaction of darunavir with ketoconazole, data were indicative for an inhibitory effect of ketoconazole on P-gp as the main mechanism for the increased transport of darunavir across the small intestine.