A new medium was developed by adding 40 mg pyrrolidonyl-beta-naphthylamide (PYR) to 1 l bile esculin (BE) agar, and was named as BE-PYR agar. To evaluate the efficacy of BE-PYR agar in differentiating enterococci from nonenterococci, we inoculated 207 strains of group D streptococci (including 157 enterococci and 50 nonenterococci) to BE-PYR agar. After incubating overnight at 35 degrees C incubator, following by applying the PYR reagent to test the color change of colonies, results indicate that the accuracy of BE-PYR agar in the differentiation of enterococci from nonenterococci reaches to 100%. Therefore, BE-PYR agar was considered to be an effective laboratory tool in further differentiation of group D streptococci.