Methods were devised for the isolation and pure culture in defined mineral medium of Closteriurn ehrenbergii Menegh (Chlorophyceae). The effect of pH, type of nitrogen source, nitrate concentration, total hardness, and various buffers on growth were investigated. Best growth rates were obtained at pH 8 to 9, but good growth was observed even at pH 9.4. Nitrate, ammonia, or urea nitrogen could be utilized, but at concentrations greater than 10-2M urea was toxic to growth. At higher concentrations of ammonia and nitrate longer lag phases of growth were obtained but eventual higher cell yields were obtained. Good growth rates were obtained with Tris-(hydroxymethyl)-aminomethae buffer at up to l0-2M; higher concentrations were inhibitory.