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The effect of mucosal application of 1mmol/l phenyl phosphorodiamidate (PPD) on the electrophysiology of isolated rumen epithelium was studied in vitro using a conventional Ussing chamber technique. Thirty-six isolated rumen epithelium samples from 5 sheep of different breeds, age and sex and solely fed on hay were used. Mucosal application of phenyl phosphorodiamidate, which has an application in improving the utilization of dietary urea in ruminants nutrition, on the mucosal side of ruminal epithelial tissues resulted in a significant (p<0.05) reduction of the transepithelial potential difference (PDt) and epithelial short circuit current (Isc), and a significant (p<0.05) elevation of the epithelial tissue conductance (Gt). Phenyl phosphorodiamidate could thus impair the barrier and transport function of the rumen epithelium.