The penicilllin-binding proteins (PBPs) of several gram-positive and gram-negative bacteria have been examined. The results indicate that: (i) PBPs are membrane proteins with molecular weights ranging from 40,000 to 120,000. When extracted with Triton X-100 from sonicated cells, they appear to fall into two patterns: one found in rods and the other in spheres. A major difference is in the low-molecular-weight component, which is usually the major PBP in bacilli but a minor one in cocci. (ii) There is a wide variation in both the number and the amount of PBPs in different bacteria, and taxonomically related bacteria tend to have similar PBP patterns. These patterns often correlate with the affinity of PBPs for penicillin and other beta-lactam antibiotics. (iii) The low-molecular-weight component usually releases penicillin spontaneously with a half-life of 10 min or less. Most, but not all, PBPs release bound penicillin in the presence of neutral hydroxylamine (0.2 to 0.8 M).