PURPOSE: To investigate the interaction of positively charged self-emulsifying oil formulations (SEOF) following aqueous dilution as a function of resulting emulsion droplet charge and size with rat everted intestinal mucosa, adherent mucus layer and Peyer's patches, using cyclosporine A (CsA) as a lipophilic model drug. METHODS: Droplet size determination (TEM technique) and zeta-potential measurements were used to characterize the resulting emulsions. For the ex vivo interaction study, the well-known rat intestine everted sac technique was used in combination with confocal microscopy. RESULTS: The positively charged oil droplets formed by SEOF dilutions at ratios of 1/50 and 1/10 elicited the stronger interaction with the mucosal surface. The positive charge of the smaller droplets was more readily neutralized, and even reversed in aqueous solutions containing moderate subphysiological mucin concentrations. Parameters such as droplet size, negativity of the epithelial mucosa potential and presence of the mucus layer on the epithelial surface affected drug mucosa uptake and the adhesion of the positively charged droplets to the rat intestinal mucosa. CONCLUSIONS: The enhanced electrostatic interactions of positively charged droplets with the mucosal surface are mostly responsible for the preferential uptake of CsA from the positively charged droplets as compared to negatively charged droplets irrespective of the experimental conditions used. The increased uptake of the CsA from the negatively charged oil droplets was consistent with the dilution extent, as expected, whereas in the positively charged droplets, an intermediate droplet size range was identified resulting in optimum drug uptake and clearly suggesting that drug uptake was not consistent with either dilution extent or droplet size.