The intestinal transport mechanism of 5-fluorouracil (5-FU) was investigated in rats using in vitro techniques, everted sacs and brush border membrane vesicles (BBMVs), and in situ perfusion technique. The gastrointestinal absorption of 5-FU was also evaluated in vivo by the analysis of gastrointestinal disposition and plasma concentration in order to examine the quantitative in vitroin situ-in vivo correlation in the absorption. In everted sacs, the uptake of 5-FU was Na⁺ and concentration-dependent, and inhibited by pyrimidines, but not by purines and nucleosides. These results are consistent with previous reports, ^3.4) and suggest the involvement of pyrimidines-specific carrier-mediated transport in the intestinal transport of 5-FU. It was also shown that the kinetic parameters were close to those in in situ perfusion. However, in BBMVs, Na⁺ dependency in the uptake of 5-FU was not confirmed. Although a slight concentration dependency and inhibition by pyrimidines were observed, the contribution of carrier-mediated transport appeared to be far smaller than that in everted sacs. Thus, it was suggested that, in addition to Na⁺, some other factor may also play an important role in the intestinal transport of 5-FU. On the other hand, oral absorption of 5-FU was shown to be gastric emptying-limited at a low dose, where the carriermediated transport is presumed to be in the linear phase (cocentration << Michaelis constant) and most efficient. The apparent membrane permeability clearance (in vivo) was in agreement with those in perfusion (in situ) and everted sacs (in vitro) at comparative concentrations. In conclusion, the carrier-mediated transport of 5-FU was confirmed to be Na⁺-dependent in everted sacs, but not in BBMVs. There may be some other factor than Na⁺ may also be required for the transport of 5-FU. It was also shown that there is a good quantitative in vitro (everted sacs)-in situ-in vivo correlation in the intestinal absorption of 5-FU.