Four different methods for defaunating were tested. Small Entodinia but some times also other species of ciliates were found on the next day after defaunation when Aerosol OT was given in doses of 5 and 10 g/animal or 1-1.5 g/kg of rumen content. Emptying and washing the reticulorumen with a 0.9% NaCl solution and subsequent freezing of the contents to -20°C for 96 h resulted in absence of protozoa for a period of 10-14 days and this was followed by the re-establishment of a population of Eudiplodinium maggii, Entodinium simplex and Dasytricha ruminantium. Emptying and washing of the reticulo-rumen with warm (40°C) water accompanied by heating of the contents to 70°C and addition of 10 g of Aerosol OT or 5 g sodium lauryl sulphate (SDS) eliminated the ciliates from the rumen for a period of 7-40 days but re-establishment of small protozoa like Entodinium simplex and Entodinium caudatum was observed later. Emptying the reticulo-rumen and washing the omasum, reticulum and rumen with warm water followed by heating of the contents to 70°C and the addition of Aerosol OT at a proportion of 1 g/kg digesta resulted in elimination of all the species of ciliates from the reticulo-rumen of all defaunated sheep for a period longer than 70 days.