A number of carbohydrates and nitrogenous adjuncts were tested for their effect on the constitutive and adaptive pentosanases produced byBacillus stibtilisandB.pumilusrespectively in a medium containing biotin, ammonium phosphate, and other mineral salts.B.subtilisproduced more enzyme with sulphite liquor than with any of the other carbohydrate sources tested. Next, in decreasing order of merit, were wheat bran, maltose, ribose, beet molasses, oat hulls, and pectin. Of the nitrogenous adjuncts tested, corn steep liquor, soybean meal, gelatin, gelysate, and ammonium lactate doubled the yield of enzyme whereas yeast extract, peptone, urea, and others were less effective. ForB.pumilusthe better carbohydrate sources, in decreasing order of merit, were wheat bran, water soluble pentosan of wheat flour, xylan, straw holo-cellulose, wheat straw, and sulphite liquor. Of the nitrogen sources, corn steep liquor was outstanding while casein, casitone, phytone, yeast extract, distillers' dried solubles, and soybean meal followed in decreasing order. A medium containing 6% wheat bran (20 mesh), 1% corn steep liquor neutralized with ammonia, 0.05% sodium chloride, and 0.05% calcium carbonate was devised for the production of pentosanase byB.pumilus. With this medium in shaken Erlenmeyer flasks, the enzyme was produced at a high rate between 12 and 40 hr.; thereafter the rate of production decreased. Maximum yields were obtained in 96 hr. A temperature of 26 °C. was more favorable for pentosanase production than higher temperatures.