Substrate utilization in defined media for two flower spiroplasmas (S. floricola and FS SR-3) and honeybee spiroplasma (HBS AS-576) was investigated. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, HBS (AS-576) could ferment trehalose; FS (SR-3), sucrose; and S. floricola, trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in growth requirements for amino acids. Only S. floricola utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and BSA were essential to all three spiroplasmas. Palmitic acid, which was non-essential, promoted growth significantly.