Prochlorococcus is the numerically dominant primary producer in open ocean ecosystems. Analysis of Prochlorococcus genome sequences from cultured isolates and ocean samples has broadened interest in studying this tiny cell, and efforts are underway to develop it into a model system for studying marine microbial ecology. A critical component of these efforts has been the development of systematic culturing methods that will facilitate the distribution of Prochlorococcus to diverse labs that may be interested in studying it. This paper provides detailed methods for maintaining cultures of Prochlorococcus, including a comparison of growth rates of cells on two artificial seawater media and on a standard medium that uses a natural seawater base. Procedures for agar plating, cryopreservation, obtaining new isolates, and issues associated with culture volume and carbon limitation also are described.