The urinary level of N-acetyl-beta-D-glucosaminidase (NAG) was reported to be a useful marker for the assessment of renal function. This enzyme was also detected at a high activity in seminal fluid. In our preliminary report, seminal NAG activity was 1,000 times higher than urinary NAG level in healthy volunteers. Moreover, the fraction ratio A to B isozymes was changed in seminal fluid independently from that in urine. In order to study the source of seminal NAG, NAG activity and the isozyme fraction were determined in the tissues of the genital organs (prostate, testis, epididymis, and seminal vesicle) as well as the urinary organ (kidney). Various levels of NAG activities were obtained, the mean tissue concentration (Unit/g protein) being 26.7 +/- 2.4, 4.3 +/- 30.5, and 8.75 +/- 0.72 unit/g, respectively, in the testis, epididymis and seminal vesicle. In particular the mean concentration of 97.7 +/- 23.3 was significantly high in the prostate. In regard to the isozyme fraction of NAG, B fraction (%) accounted for 15.1 +/- 5.5, 33.6 +/- 8.2, 37.7 +/- 0.1, and 70.6 +/- 4.5 percent, respectively, in the testis, epididymis, seminal vesicle and prostate. It was to note that the A to B fraction ratio in the prostate corresponded to that in the seminal fluid. These results suggest that seminal NAG is mainly from the prostate.