Abstract.Purification andin vitrocultivation techniques were developed for the fish haemoflagellate,Trypanosoma danilewskyi.The parasites were isolated and purified from the peripheral blood of experimentally infected goldfish, using a combination of Ficolt‐paque gradient centrifugation to remove fish red blood cells andin vitroincubation to remove the remaining fish leucocytes. A serum‐free culture medium forT. danilewskyisupported both short‐ and long‐term cultivation of the haemoflagellates. The serum‐free medium is a mixture of reagents available commercially: Leibovitz's L‐15 medium, Dulbecco's Modified Eagle Medium and Hank's balanced salt solution. The doubling time was calculated to be 44.4 × 7.8h. Typically, a two‐ to five‐fold increase in the number of cultured parasites was observed on day 7 after subculture with 1 × 106and 5 × 105trypanosomes ml−1, respectively. When administered to fish, thein vitro‐derived parasites caused an infection and pathology whose characteristics were similar to those observed following infection with trypanosomes obtained from infected goldfish. The freshly isolated andin vitro‐grown parasites were successfully cryoprescrvcd in the culture medium containing 10% glycerine at −80°C for at least 3 months. Although the viability of the parasite decreased by 40–50% after thawing, cryoprcserved parasites retained the ability to infect goldfish.Correspondence: Dr M. Belosevic, Associate Professor, Department of Zoology, CW‐312 Biological Sciences Building, University of Albert