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BALB/cBYJ mice were immunized against larval Onchocerca volvulus by subcutaneous injection of normal, irradiated, or freeze-thaw-killed Onchocerca sp. larvae. The mice received challenge infections of O. volvulus third-stage larva (L3) contained in diffusion chambers implanted subcutaneously. At two-weeks postinfection, the diffusion chambers were removed and larval survival was assessed. When mice were immunized a single time with 35-krad-irradiated or normal O. volvulus L3, there was a significant reduction in the survival of challenge parasites. However, there was little or no reduction in challenge worm survival when mice were immunized a single time with freeze-thaw-killed O. volvulus L3 or fourth-stage larva (L4), or irradiated O. lienalis L3. When a second dose of freeze-thaw killed O. volvulus L3 or irradiated O. lienalis L3 was administered, there was a significant reduction in parasite survival in immunized mice. Immunization with O. volvulus L4 or a combination of L3 and L4 failed to confer protection. These results demonstrate that mice can be immunized against larval O. volvulus and that diffusion chambers are an efficient method for studying protective immunity to this parasite in a mouse model.