The antigenic relationships of hyaluronidases, bound and free, associated with temperate bacteriophages of group A streptococci were examined with antibody against purified whole phage and with antibody against phage-bound enzyme released by urea and purified to homogeneity. Studies performed by double diffusion in agar (ouchterlony) with antibody against the homologous purified enzyme from a temperate phage of a type 49 streptococcus indicated that the bound and free enzyme gave a single line of identity and that the free hyaluronidase activities in induced lysates of four strains of M type 49 streptococci were immunologically indistinguishable but different from the enzyme in induced lysates of a heterologous type. The four M type 49 strains were from widely different geographical or temporal sources and of different phage subtypes as determined by lyxic patterns. These findings were confirmed in studies that employed a functional assay of enzyme neutralization. An immunoglobulin preparation of antiserum against the purified enzyme as well as one against homologous purified whole phage neutralized the hyaluronidase activity produced by induction of the M type 49 strains and present either phage-bound or soluble in phage-free lysates. These immunoglobulin preparations had little effect on the hyaluronidase activities present in phage-lysates of other M types of group A streptococci. Inhibition of propagation of temperate phages by antibody against the purified phage hyaluronidase paralleled the neutralization of phage-associated enzyme activity by this antibody, indicating that antibody to the purified enzyme can inhibit phage infection. Antibody preparations against the purified phage-bound enzyme or against purified whole phage did not neutralize the extracellular hyaluronidase in the supernate of an uninduced culture of M type 4 streptococci. A human serum strongly inhibitory for the extracellular enzyme of this strain or on the purified phage enzyme from an M type 49 strain. The results support the view that the hyaluronidases associated with the temperate bacteriophages from various M types of group A streptococci do not share common antigenic determinants but that an immunological specificity exists that parallels the serologic specificity of the M protein of the host strains.