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. Most, if not all, choanoflagellates are heterotrophs that prey on bacteria. Thus, all choanoflagellate media provide nutrition for bacteria, which are in turn consumed by the choanoflagellates. If a culture is axenic or has low bacterial content, it can be supplemented with a specific bacterial strain that has been grown separately. On the other hand, if a culture already contains a well-established flora of bacteria, it can be cocultured with the choanoflagellate isolate. In either case, it is common to enrich the medium with an organic extract, such as liver extract, cereal grass infusion, or a mixture of proteose peptone and yeast extract. This protocol describes how to start cultures of the marine choanoflagellate species, Monosiga brevicollis, from frozen stocks. These cultures can then be maintained and expanded in preparation for DNA or RNA isolation or cell biological assays.