Urinary N-acetyl-beta-D-glucosaminidase (NAG) is an early physiological indicator of renal damage in several mammalian species. A study was conducted to confirm occurrence of NAG in hen urine, to establish baseline urinary NAG in laying hens, and to assess the feasibility of using the enzyme as a marker of renal damage in hens. Hy-Line(R) hens were used in a completely randomized block design in the first part of the study. Urine was collected at 4 to 6, 6 to 10, 10 to 14, and 14 to 18 h, and serum at 4, 6, 10, and 14 h postoviposition, and assayed by spectrophotometry for NAG. Kidney tissue from additional hens was assayed histochemically for NAG. Serum NAG (range: 0.11 to 0.14 mU/mg protein) was found to be several orders of magnitude lower than urine NAG (6.44 to 12.27 mU/mg protein). Urine NAG increased from 4 to 6 h through 14 to 18 h, indicating that time of collection is critical in order to utilize the enzyme as a valid marker for laying hens. A preliminary study with five hens indicated that 10 d of treatment with liquid cholecalciferol (D-3) supplement (three times the recommended level) were not enough to detect renal damage on the basis of significant changes in urine NAG, but elevated urine NAG was detected at 40 d of D-3-supplementation. Overall the results indicate that NAG in urine of laying hens is a potentially useful diagnostic marker of renal damage.