1. Intestinal xenobiotic transporters are a significant barrier to the absorption of many orally administered drugs. P-glycoprotein (PGP) is the best known, but several others, including members of the multidrug resistance-associated protein (MRP) family, are also expressed. Definitive information on their precise effect on intestinal drug permeability is scarce due to a lack of specific inhibitors and the difficulty of studying non-PGP activity in the presence of high PGP expression. 2. We have investigated the in vitro use of intestinal tissues from PGP knockout (mdr1a (-/-)) mice as a tool for dissecting the mechanisms of intestinal drug efflux. The permeability characteristics of digoxin (DIG), paclitaxel (TAX) and etoposide (ETOP) were measured in ileum from mdr1a (-/-) and wild-type (FVB) mice mounted in Ussing chambers. 3. DIG and TAX exhibited marked efflux across FVB tissues (B-A : A-B apparent permeability (P(app)) ratio 10 and 17 respectively) which was absent in mdr1a (-/-) tissues, confirming that PGP is the sole route of intestinal efflux for these compounds. The A-B P(app) of both compounds was 3 - 5 fold higher in mdr1a (-/-) than in FVB. 4. Polarized transport of ETOP in FVB tissues was reduced but not abolished in mdr1a (-/-) tissues. Residual ETOP efflux in mdr1a (-/-) tissues was abolished by the MRP inhibitor MK571, indicating involvement of both PGP and MRP. 5. MK571 abolished calcein efflux in mdr1a (-/-) tissues, while quinidine had no parallel effect in FVB tissues, suggesting involvement of MRP but not PGP. 6. Tissues from mdr1a (-/-) mice provide a novel approach for investigating the influence of PGP ablation on intestinal permeability and for resolving PGP and non-PGP mechanisms that modulate drug permeability.