Polymorphism of hyaluronidase (EC 3.2.1.35) was detected in the serum from 6 out of 20 vertebrate species by electrophoretic analysis. Electrophoresis was performed on a hyaluronan-containing polyacrylamide gel, that visualizes hyaluronidase activity upon incubation at acid pH. No hyaluronidase activity was detected in the sera of horse, swine, cattle, goat, sheep, rabbit, chicken, Triton alpestris, Triton palmatus, Triton vulgaris, pleurodeles, axolotl, eel and dog-fish. The 6 positive sera were from man, mouse, rat, Syrian hamster, dog and Triton cristatus. In each of these species, the electrophoretic banding pattern of hyaluronidase was different, as was the activity per unit volume of serum. Furthermore, in mice, the 12 strains analyzed could be divided into 3 groups, containing the following numbers of hyaluronidase bands; 8 (BALB/c/J, BALB/c/By, ICFW, SW, XVIInc/Z), 5 (DBA/2 Mrc Ico, DBA/2 Mrc Ico nu/nu, B10.D2/nSn), and 1 (C57B/Rho Ico, C57BL/6/By, C57BL/6/J Ico, C57BL/6/J Ico nu/nu). Human serum generally displayed only 1 band, although there was a 2nd faint band in a few cases and a 3rd in 1 case. Rat serum displayed 4 bands, Syrian hamster serum, 3, and dog and Triton cristatus serum, 1.