Hyaluronidase activity was detected and partially characterized in salivary gland extracts of females of six sand fly species. In Phlebotomus papatasi and Lutzomyia longipalpis the enzyme was active over a broad pH range; the pH optimum was 5.0. Besides high cleaving activity towards hyaluronic acid, it hydrolyzed chondroitin sulfates A and C. Hyaluronidases of various sand fly species differed in structure and sensitivity to reducing conditions. In the subgenera Phlebotomus (P. papatasi and P. duboscqi) and Adlerius (P. halepensis) the predominant active form of the enzyme was monomeric with the same apparent molecular weight under nonreducing and reducing conditions (around 65 kDa for P. papatasi and P. duboscqi and 110 kDa for P. halepensis). In P. sergenti the enzyme occurred as a putative homodimer but remained active under reducing conditions when separated into 60 kDa subunits. In L. longipalpis and P. perniciosus the activity was detectable under non-reducing conditions only. In P. duboscqi, low enzyme activity was found also in males. Salivary gland hyaluronidases of sand flies share characteristics with endo-N-acetyl-hexosaminidases of mammalian sperm cells and corresponding venom enzymes of Hymenoptera. Hypothetically, they facilitate blood meal acquisition but also may modulate immune reactions of the host and promote pathogen transmission.