In a prospective study the prevalence of high-level aminoglycoside resistance (MIC greater than or equal to 2,000 micrograms/ml) among 62 clinically significant enterococci was investigated. A total of 10(5) organisms were inoculated a) onto a plate containing 2,000 micrograms/ml of gentamicin or streptomycin; b) into a microtube for dilution MIC determinations for gentamicin, amikacin, tobramycin and streptomycin; and c) into a single tube containing 500 micrograms/ml of gentamicin, amikacin, tobramycin or streptomycin in supplemented Mueller-Hinton broth. In addition, tubes containing 500 micrograms/ml of gentamicin, amikacin, tobramycin or streptomycin were inoculated with five enterococcal colonies ("crude" method). For 45 of the 62 isolates, MICs of gentamicin, amikacin and tobramycin were less than or equal to 500 micrograms/ml, while 17 (27%) showed high-level resistance. The MICs of streptomycin were less than or equal to 500 micrograms/ml for 42 of 62 isolates, and greater than or equal to 2,000 micrograms/ml for 20 (32.3%). For 8 of the 17 (47%) isolates showing high-level gentamicin resistance, MICs of streptomycin were less than or equal to 500 micrograms/ml. There was complete agreement between the results of the plate method, the microtube dilution MIC and the tube inoculated with 10(5) CFU, but the crude method gave discordant results for two isolates. It is concluded that a tube containing 500 micrograms/ml of aminoglycoside is a simple, accurate and inexpensive method for determining high-level aminoglycoside resistance.