Salivary gland extracts of several Simulium spp. were shown to contain vasodilative activity as measured by the rapid and persistent induction of erythema in response to intradermal injection into rabbit skin. Total salivary gland activities were approximately equal for S. vittatum, S. metallicum, S. bivittatum, and S. argus (titers of 0.03–0.02 pairs of gland). Total gland activity in the highly anthropophilic species S. ochraceum, however, was an order of magnitude greater, with erythema produced by as little as 0.002 pairs of glands. Tests for physical stability of the activities from two species (S. vittatum and S. ochraceum) indicated that the vasodilators were proteinaceous and heat stable. A two-step, reversed-phase high-performance liquid chromatography (HPLC) procedure was developed that isolated both activities with similar elution patterns. Homogeneity of the purified protein from S. vittatum was confirmed by capillary gel electrophoresis. Electrospray ionization mass spectroscopy of the S. vittatum protein detected a mass of 15,351 daltons. Similarity in elution times of the proteins from a TSK HPLC column predict some structural similarities between the S. vittatum and S. ochraceum vasodilator proteins.