The unstirred water layer (UWL), which has been known to exist in the boundary of the intestinal lumen and intestinal wall, often behaves as an absorption barrier especially for lipophilic drugs. The intestinal absorption of drugs is often characterized using Caco-2 cell monolayers grown on Transwell polycarbonate membranes. The permeability $(P_{app})$ of drugs across the cell monolayer might be influenced by the agitation of the donor compartment, since the width of UWL on the surface of the cell monolayer would be reduced by the agitation. In this study, the effect of agitation of the donor compartment with 60 rpm on the permeability was measured for 12 drugs with a wide range of lipophilicity and permeability. The $P_{app}$ of mannitol, tributylmethyl ammonium, cimetidine, ranitidine, hydrocortisone, benzylpenicillin and loxoprofen was not influenced by the agitation, while the $P_{app}$ of theophylline, propranolol, YH439, phenylpropanolamine and testosterone was increased by the agitation. There was a significant correlation between the increase of $P_{app}$ by agitation and the lipophilicity for the compounds having $P_{app}>2{ imes}10^{-5}$ cm/sec. No correlation was observed for the difference in $P_{app}$ by agitation and the molecular weight, or lipophilicity of the drugs. Therefore, the agitation rate of the donor compartment in the Caco-2 cell monolayer study should be carefully controlled in order to estimate $P_{app}$ reproducibly especially for lipophilic drugs.