The methylxanthines caffeine, theophylline, and isobutylmethylxanthine greatly increased spore yields of Clostridium perfringens strains FD-1, PS52, and PS49 when grown on Duncan-Strong medium or on a new casein-digest medium. Four other strains (KA3, and National Collection of Type Cultures strains 8798, 8238, and 10240) failed to show any significant increase when tested under similar conditions. The degree of sporulation increase was influenced by the carbohydrate energy source in some strains but not in others. Strain PS52 showed a large increase in spore yield when dextrin was the energy source but only a slight increase when raffinose served as the energy source. Strain FD-1 showed similar increases in spore yield with either dextrin or raffinose. The difficulty commonly associated with inducing sporulation of Clostridium perfringens on laboratory media (22) has impeded studies on the spores of this important organism for many years. The most commonly employed sporulation medium for C. perfringens is probably that of Duncan and Strong (6). Although satisfactory spore yields (>107/ml) have been obtained on this medium with some strains, many strains sporulate only to a very limited extent on it. Ellner's medium (7) and caseindigest media (10) have also been used (3, 4, 8, 9, *15; H. Riemann, Ph.D. thesis, University of Copenhagen, Denmark, 1963), but none are consistently satisfactory (22). Recently we reported that methylxanthines could greatly increase spore yields of strain PS49 in a defined medium and in a new caseindigest medium (14). In the present report, we show that strains PS52 and FD-1 also exhibited large increases in spore yield when methylxanthines were incorporated with the casein-digest medium. Strains PS49, PS52, and FD-1 also demonstrated very significant increases in spore yield when methylxanthines were added to Duncan-Strong (DS) medium. Four other strains showed no increase in spore yield when tested under similar conditions. MATERIALS AND METHODS Medium. The basal medium (CPS) contained Casitone (Difco) (6%), K2HPO4 (0.1%), ethylenediaminetetraacetic acid, ferric salt (0.02%) (11), and