Exsheathed larvae of Haemonchus contortus were frozen over liquid nitrogen, and were kept frozen for 44 weeks. Upon thawing, the larvae showed no loss in viability as judged by motility. Their infectivity to sheep was compared with that of normal larvae (ensheathed and nonfrozen). Sheep given 1,500 frozen exsheathed larvae acquired worm counts (688 to 921, mean 722) similar to those given normal larvae (439 to 852, mean 627). It has been reported from this laboratory that exsheathment of the infective larva of the nematode Haemonchus contortus enables it to withstand freezing in the presence of liquid nitrogen (Campbell et al., 1972). When that report was prepared for publication the longest survival time recorded was 4 weeks; and although the larvae had been shown to be highly infective, it had not then been possible to make a critical comparison of the infectivity of frozen and nonfrozen larvae in worm-free sheep. The present study shows that frozen larvae remain viable (without perceptible loss in numbers) for at least 44 weeks and that their infectivity is unimpaired. MATERIALS AND METHODS Use was made of a strain of H. contortus originating on Branchburg Farm, New Jersey, and maintained by serial passage in sheep for many years. Larvae were harvested from conventional fecal cultures after 7 days' development at 27 C and approximately 90% relative humidity. The larvae of this strain can readily be exsheathed by bubbling carbon dioxide into the suspension of larvae, followed by agitation at 37 C; and this was the method employed for exsheathment. The larvae were concentrated in "Vitro" brand 1-dram screw-cap vials (Wheaton Glass Company, Milville, New Jersey). Each vial contained roughly 150,000 larvae in 1 ml water. They were frozen by transferring the vials directly from air at room temperature to the storage section of a liquid nitrogen container (Union Carbide Corporation, Model Designation Linde LD-40). According to the manufacturer of the unit, the temperature in this sealed space would not be more than 96 C higher than the temperature of the underlying liquid nitrogen (-196 C). The vapor space contained 4 layers of steel storage drawers; the vials were stored in the second layer from the top, but we Received for publication 20 October 1972. have no reason to believe that the exact location was a critical factor. The larvae were thawed by immersing them in water at about 55 C, allowing approximately 300 ml of water per vial, and making no attempt to maintain the initial temperature of the bath. As soon as the contents of the vial were thawed, the larval suspension was diluted several-fold with water at room temperature and allowed to stand at room temperature for 1 hr before the motility was assessed. The lambs used in the infectivity trial had been raised on slatted floors which were cleaned daily, and were kept under these conditions throughout the experiment. They were known to harbor light infections of Capillaria, Strongyloides, and Eimeria spp. at the time of use, but were not passing trichostrongyle eggs in the feces. The lambs were of both sexes and were approximately 3 months old. At necropsy the abomasum was removed and the contents collected on a 200-mesh sieve (pore size 0.074 mm). The contents were stained with iodine and examined in their entirety to determine the total number of worms present. The abomasum was allowed to soak in saline at 37 C for 24 to 48 hr, with the addition of an antibiotic preparation to suppress putrefaction. The resultant material was collected on a 200-mesh screen, stained, and examined in its entirety for worms. EXPERIMENTS AND RESULTS Larvae of H. contortus were exsheathed and frozen on 25 August 1971. A sample of these larvae, henceforth designated "frozen" larvae, was removed from the container on 28 June 1972, that is, after 44 weeks in the frozen state. After thawing, examination of 200 larvae under a stereoscopic microscope revealed that 92% of them were motile. It is possible that an even greater percentage of the larvae in this culture would have been motile prior to freezing; but the motility of 88% of the larvae in the nonfrozen control culture (below) is