A simple plate assay for hyaluronidase activity in biological samples is described. Hyaluronic acid is incorporated into agarose gels and the enzyme is allowed to diffuse from punched wells. The undigested hyaluronic acid is then precipitated with cetylpyridinium chloride and the diameters of the clear circles are proportional to the logarithm of the enzyme concentration applied to the well. The assay was utilized to examine commercially available hymenoptera venoms, manufactured for use in allergy diagnosis and treatment, for their content of hyaluronidase as a measure of lot-to-lot consistency. The assay permits the analyses of a large number of samples with good reproducibility, without the need for any special instrumentation. Based on the quantity of purified hyaluronidase reported in honey bee venom (T. P. King, A. K. Sobotka, L. Kochoumian, and L. M. Lichtenstein, 1976, Arch. Biochem. Biophys.172, 661–671) we estimate that the assay should detect 70 ng/ml of purified honey bee venom hyaluronidase.