Treatment with aminoglycosides is known to cause irreversible hearing loss, typically affecting higher-frequency hearing first and progressing to lower frequencies. Standardized methodology has not been developed for early detection of ototoxicity. Serial conventional (0.25-8 kHz) and high-frequency (9-20 kHz) hearing threshold monitoring was done prospectively in 53 hospitalized patients administered aminoglycosides. Hearing loss occurred in 47% of the ears studied, with hearing loss first appearing in the high-frequency range in 71% of ears showing change. Analysis of data on an individual basis revealed a five-frequency range most susceptible to initial ototoxicity. Testing only this range would have resulted in early identification of 82% of ears showing change. Results confirm the critical need for serial auditory threshold monitoring encompassing high frequencies in patients receiving aminoglycosides. A shortened five-frequency monitoring protocol is presented and suggested for use with patients unable to tolerate lengthy audiometric testing procedures.