In recent years, a growing number of reports have suggested that the protozoan parasite, Blastocystis hominis (Brumpt 1912), is a pathogenic cause of diarrhoea. It is imperative, however, that more studies are conducted on the various human isolates of the parasite before incriminating it is a pathogen. In our laboratory we maintain human and animal isolates of Blastocystis using the monophasic medium Iscove's Modi®ed Dulbecco Medium (IMDM) as established by Ho et al. (1993), but maintaining continuous cultures is tedious, time consuming and expensive, and the parasites may be prone to genetic drift. There is therefore a need to cryopreserve these isolates before biochemical, molecular and other biological studies can be undertaken. However, very little is known about the cryopreservation of this parasite.The cryopreservation protocol described by Zierdt (1991), who used dimethylsulphoxide (DMSO) to cryopreserve Blastocystis, demanded extreme care, especially during the process of slow cooling and the subsequent maintenance of parasites in liquid nitrogen. In our previous study (Suresh et al., in press), we showed that the cryoprotective ability of glycerol and mannitol to preserve Blastocystis was enhanced when DMSO was added. We used a protocol which was simpler than that described by Zierdt (1991), but the parasite recovery from this cryopreservation medium upon reculture was low, despite an initially high parasite concentration (200´10 6 ml). The original culture medium had to be supplemented with 20% horse serum to successfully recover cryopreserved parasites after deep freezing.The present paper reports for the ®rst time a protocol for the cryopreservation of Blastocystis using fetal calf serum. The medium was prepared by adding 3.5 ml fetal calf serum (Gibco) to 7.5 ml glycerol and 89 ml minimum essential medium (MEM). The fetal calf serum used was non-heat-inactivated serum. The medium was then dispensed into 20-ml universal bottles and stored at A20°C until use.An axenic culture of B. hominis (isolate C), previously obtained from a Blastocystis-infected patient was continuously maintained in IMDM (Gibco) with 10% horse serum in anaerobic chambers at 37°C (Ho et al. 1993). Parasites, mostly vacuolar forms from anaerobic cultures of axenic isolate C, were centrifuged and the sediments containing the parasites were pooled. The concentration was made up to 50´10 6 , 100´10 6 and 200´10 6 of vacuolar forms in IMDM.Twenty preparations of each parasite concentration were placed in 2-ml cryovials (Nunc) containing the above-mentioned medium and were then allowed to stand at room temperature for 30 min. They were then placed in a polystyrene box and transferred to a A20°C freezer for 2 h. Polystyrene boxes containing these cryovials were then stored at A70°C overnight before transfer to liquid nitrogen for storage.Batches of cryovials were maintained for 2 and 8 weeks in liquid nitrogen and then thawed at 37°C in a water bath for 1.5±2 min. The cryovial contents were quickly transferred under sterile conditions t...