Infective larvae (L3) of Onchocerca volvulus were procured in Liberia, West Africa, in the natural black fly vector, Simulium yahense. A cryobiological technique was developed to preserve L3 of O. volvulus that were fully viable after thawing. Larvae were treated before cooling with 4 cryoprotective compounds. Three compounds, dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol, were prepared with distilled water. The fourth compound was DMSO prepared in different concentrations with 0.25 M sucrose. The treatment with DMSO + 0.25 M sucrose cryoprotectant resulted in the highest survival of infective larvae. Five cooling rates between 0.5 C/min and 20.0 C/min were applied. The highest survival of L3 was with the cooling rate of 1.0 C/min. Two-step cooling of L3 was applied. In the first step, L3's were frozen to 5 levels from -10.0 C to -20.0 C, -30.0 C, -40.0 C, -60 C, and -80.0 C, and in the second step, larvae were transferred into liquid nitrogen at -196 C for rapid cooling and storage. The survival was the highest when larvae were cooled to approximately -40 C prior to transfer into liquid nitrogen. Slow, gradual, and rapid thawing procedures were applied. The survival was the highest in rapid warming.