Third-stage larvae (L3) of the human hookworm parasite Ancylostoma ceylanicum were cultured from the feces of infected hamsters and frozen for up to 100 days in liquid nitrogen. Upon thawing, viable larvae were recovered and used to inoculate naive hamsters. The larvae recovered from this second group of hamsters were used to inoculate a third group of naive animals, which demonstrated that the originally frozen larvae were successfully maintained for two full generations following thawing. These data suggest that exsheathment, which has previously been reported to be essential for successful cryopreservation, is not necessary for recovery of viable, infectious A. ceylanicum L3.