Tetragonal layer protein (T-layer) isolated from Bacillus sphaericus NTCC 9602 (wild type) or 9602 Lmw (variant) bonded specifically to the sacculi (peptidoglycan) of either cell type. Only uncleaved T-layer subunits were capable of specific recognition of the B. sphaericus sacculi; other Bacillus strains and gram-positive bacterial sacculi would not adsorb B. sphaericus strain 9602 T-layer. The peptidogylcan did not function as a template since isolated T-layer subunits self-assembled into characteristic pattern. Upon reassociation with sacculi, T-layer assemblies were randomly oriented patches compared with more continuous strictly oriented pattern on cells or fresh cell walls. T-layer associated with the sacculus was less susceptible to conditions that dissociated in vitro-assembled T-layer. Mild proteolysis of both wild-type and variant T-layer subunits by a variety of enzymes reduced the molecular weight by 18,000 in all cases, indicating that one region of the molecule was particularly susceptible to cleavage. Subunits from which the minor fragment had been cleaved upon aging retained the capacity to assemble in vitro, but would no longer adsorb to sacculi. Thus, the ability of T-layer to form networks was separate from its ability to bind cell walls, and the 18,000-dalton piece of the T-layer polypeptide was necessary for attachment to the cell wall.