The effect of low temperatures on the viability of rumen protozoa was studied. In Trial 1, samples of mixed rumen contents were diluted and cultured at 38, 15 and 5°C for 2, 4 and 6 h, respectively. Viability of Isotrichidae was not affected by either the length of incubation or the temperature (P < 0.10). Incubation at low temperatures negatively affected the viability of Entodinium, Diplodiniinae and Ophryoscolex. Differences between 15 and 5°C were not significant except for Entodinium spp. (P = 0.07). The reduction of viability increased with time, this effect being noticeable (P < 0.10) for Entodinium (after 4 and 6 h), Diplodiniinae (at 6 h) and Ophryoscolex (at 2 and 6 h). Preservation of mixed rumen protozoa for at least 4 h at 15 or 5°C did not markedly affect viability, suggesting that cultures can be recovered after short-term refrigeration. In Trial 2, monocultures of Entodinium caudatum and Diploplastron (Metadinium) affine were cultured at 5°C for 4, 8 and 24 h with or without milk powder/egg yolk solution as a membrane protector. For both species, viability was not affected after 4 h at 5°C, but decreased from 8 h onwards (P < 0.01). The use of a membrane protector improved viability of D. affine at all time periods (P < 0.05), but only at 24 h for E. caudatum (P = 0.07), showing that the use of membrane protector may be positive at medium or long exposure to low temperatures, depending on the species.