A method has been developed for the isolation of chloroplasts from Euglena gracilis grown under mixotrophic conditions. This method utilizes sucrose density-gradient centrifugation in the AXII zonal rotor and allows the rapid preparation of large amounts of chloroplasts free from contaminating whole cells and other cytoplasmic materials. The majority of the isolated chloroplasts appear intact in phase contrast and electron micrographs. The purified chloroplast fraction contains DNA, the major species of which has a density of 1.682 g/cm(3). The species of DNA having a density of 1.707 g/cm(3) seemed to result from the presence of contaminating nuclear fragments which could be removed by isopycnic flotation.