Aims:  To study the viability of a culture of the rumen protozoon Entodinium caudatum after a cryopreservation procedure by a fluorescence microscopy staining method. Methods and Results:  Fluorescence method is based on the different colour of cells depending on their membrane integrity. When the temperature effect was studied either by fluorescence or motility, the techniques were correlated (r = 0·727) and their slopes and intercepts were not different (P > 0·05). However, motility showed a higher variation coefficient (0·40 vs 0·12). There were no differences between cooling rates at cryopreservation (1 and 4°C min−1) at 38, 15 or 5°C, nor after thawing. Conclusions:  Fluorescence staining is more accurate than motility for assessing protozoal viability. Viability after thawing was 0·50, and the number of viable cells per 250 μl straw was 320 and 420 for 1 and 4°C min−1. Significance and Impact of the Study:  This cryopreservation procedure seems to ensure culture recovery for E. caudatum.