The activity of the urinary enzymes alanine aminopeptidase (AAP; EC 3.4.11.2) and N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) was measured after eliminating distorting factors in ten healthy dogs on three consecutive days in order to determine inter- and intra-individual variability. All the animals were being housed and fed in the same way. The urine (2nd morning urine) was collected between 8:30 and 10:00 a.m. by means of ultrasound-controlled cystocentesis. Our comparative measurements of native and gel-filtered urine showed an increase in enzyme activity in all of the samples in the case of AAP and in most of the samples in the case of NAG, thus proving the existence in the dog of AAP and NAG urinary enzyme inhibitors. The large inter- and intra-individual AAP and NAG fluctuation ranges were reduced considerably by relating enzyme activity to urine creatinine concentration. The provisional upper limit of the AAP reference range is 6.3 U/g creatinine (90% percentile), that of NAG 6.2 U/g creatinine (90% percentile). The AAP and NAG enzyme activities remained constant in gel-filtered samples kept at 4 degrees C for at least 5 days, in those kept at -18 degrees C for at least 4 weeks.