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The chemistry of the binding of14 C-benzylpenicillin to sporulating cultures ofBacillus megaterium andB. subtilis is similar to that in a 4-hr vegetative culture ofStaphylococcus aureus . Unlabeled penicillins prevent the binding of14 C-benzylpenicillin, but benzylpenicilloic acid and benzylpenilloic acid do not. Bound antibiotic can be removed from cells with neutral hydroxylamine at 25 C. Sporulating cultures display two intervals of enhanced binding, whereas binding to stationaryphaseS. aureus cells remains constant. The first period of increased binding activity occurs during formation of the spore septum or cell wall primordium development, and the second coincides with cortex biosynthesis.