Primary isolation of Prototheca was accomplished on MacConkey's, sheep blood agar, and Sabouraud dextrose agar. Prototheca was differentiated from similar organisms by its ability to grow on MacConkey's agar, and by its colonial morphology. Further differentiation was based on staining procedures to reveal the characteristic microscopic morphology. In the process of identification of several isolates of Prototheca zophii obtained from animal sources, a simple guideline for isolation and identification of these organisms was developed.