The penicillinases of Bacillus licheniformis, Bacillus cereus, and Staphylococcus aureus are related in structure and cellular localization to one another more closely than they are to penicillinases from Gram-negative organisms. In the latter, penicillinases are almost exclusively found in the periplasm, while the Gram-positive bacteria retain a substantial proportion as hydrophobic membrane-bound forms. We recently showed (Nielsen, J. B. K., Caulfield, M. P., and Lampen, J. O. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3511-3515) that B. licheniformis membrane attachment was achieved through a glyceride thioether modification identical to that in several Gram-negative outer membrane proteins. We now report that the membrane penicillinases of S. aureus and B. cereus also possess the modification. We do this by demonstrating isotopic labeling of these forms of [3H]palmitate, by showing that they exhibit the same response to the antibiotic globomycin which appears to inhibit processing steps specifically involving the glyceride thioether in Escherichia coli outer membrane proteins, and lastly by isolating glyceryl cysteine sulfone, the oxidation product of the modified cysteine residue. By comparing the modification-susceptible signal sequences of Gram-positive penicillinases and of Gram-negative outer membrane proteins with those of nonmodified Gram-negative penicillinases, we describe in increased detail the structural features within the signal sequence that allow modification and cleavage resulting in membrane anchorage.