Penicillins F, G, and X (English designation I, II, and III, respectively), which often occur in widely differing proportions in various samples, possess different activities toward Staphylococcus aureus NRRL B-313 (FDA strain 209 P), and Bacillus subtilis NRRL B-558 (both smooth and rough phases) when assayed by the cylinder-plate method. When pure Na penicillin G (0.6 7 = one unit) is used as a standard, pure Na penicillin F has an activity of 1490 units per mg. against Staph. aureus and 970 units per mg. against B. subtilis (either S or R phase). Similarly, pure Na penicillin X has an activity of 845 units per mg. against Staph. aureus, 800 units per mg. against B. subtilis B-558S, and 1200 to 1700 units per mg. against B. subtilis B-558R, the latter value presumably being dependent upon the variation in the degree of dissociation of B. subtilis Such differential assays may serve as the basis for calculating the approx. composition of pencillin mixtures comprised of these 3 entities. The importance of these observations with respect to the use of B. subtilis as the sole test organism, and the significance of strain and phase susceptibility to antibiotic compounds are discussed.