INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Choanoflagellates can be stored under stable conditions by freezing them with liquid nitrogen, as described here. To prevent cross-contamination among vials stored in the liquid phase of a liquid nitrogen freezer, the use of CryoFlex tubing to fully seal the vials is encouraged.