A water-soluble metabolite, isolated from the urine of dogs given (S)-5-ethyl-5-phenylhydantoin [(S)-EPH], has been identified as 1-deoxy-1-[(5S)-5-ethyl-5-phenylhydantoin-3-yl] beta-D-glucopyranuronate [(S)-EPH N-glucuronide]. EPH N-glucuronide did not release the aglycone upon acid or beta-glucuronidase treatment, but incubation in alkaline solution (pH 12-13) readily formed 2-ethyl-2-phenylhydantoic acid (EPHA). The EPHA so formed could be quantitatively cyclized to EPH. With the knowledge of the conversion efficiency of EPH N-glucuronide to EPHA, a quantitative GLC assay for the metabolite was developed. EPH N-glucuronide was found to be the major urinary metabolite after administration to dogs of either (R)-, (S)-, or (RS)-EPH.