Cryopreservation is a practical method for stabilizing the genetic content of living algae over long periods of time. Yet, Chlamydomonas reinhardtii, the algal species most often utilized in studies requiring genetically defined strains, is difficult to cryopreserve with a consistently high post-thaw viability. Work described here demonstrates that C. reinhardtii retains high viability only when cryopreserved at a low cell density. Low viability at high cell density was caused by the release of an injurious substance into the culture medium. Rapid freezing and thawing under non-cryoprotective conditions released large amounts of the injurious substance. Heat denaturation of cells prevented the release of the injurious substance, but heating did not inactivate it after it was released. Even when concentrated, the injurious substance was non-toxic to cells under normal culture conditions. Reduced viability of cells cryopreserved in the presence of the injurious substance could not be attributed to changes in the tonicity of the medium. A mutant strain of C. reinhardtii (cw10) with a greatly diminished cell wall did not release a substance that reduced the post-thaw viability of wild-type or cw10 cryopreserved cells. Cryopreservation of cw10 cells was achieved with approximately the same post-thaw viability irrespective to the cell concentration at the time of freezing. Acid treatment of the injurious substance was able to partially diminish its injurious effect on cells during cryopreservation. We propose that diminished viability of C. reinhardtii cells cryopreserved at high cell densities is caused by the enzymatic release of a cell-wall component.