We evaluated the effect of three variables (cryoprotectant temperature, light regime, and time of exposure to the cryoprotectant) throughout the equilibrium period during cryopreservation on the viability of the microalga Chaetoceros calcitrans (Paulsen). For this, the cryoprotectant dimethyl sulfoxide (DMSO) at 5% (v/v) was added at three different temperatures (4, 10, and 25°C) before placing the microalgae in cryobiological straws for freezing. Once inside the cryobiological straws, the microalgal-cryoprotectant suspensions were subjected to the following light regimes for 15 or 45 min: complete light, complete darkness, light/darkness, and darkness/light. Suspensions were then frozen under controlled conditions and stored in liquid nitrogen. The viability index proposed by Cañavate & Lubian (1995b) was used to measure microalgal viability after cryopreservation. Results indicated that it is necessary to use a cryoprotectant to ensure the viable cryopreservation of C. calcitrans. Statistical analyses showed that the temperature of the DMSO influenced the viability of cryopreserved microalgae and that there was no synergistic effect between the other variables studied. The viabilities obtained with DMSO at 25°C, 10°C, and 4°C were 34.9%, 27.8%, and 20.6%, respectively.