Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75° C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80° C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45° C. After thawing, atrial cells showed 53 ± 5% of the metabolic activity, 84 ± 5% of the number, and 92 ± 2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25° C yielded the best results. The thawed ventricular cells showed 83 ± 5% of the metabolic activity, 91 ± 5% of the number, and 96 ± 2% of the viability of nonfrozen cells.