We have analyzed the location of the epitope areas of the four monoclonal antibody groups against penicillin-binding protein 1B (PBP 1B; T. den Blaauwen, F. B. Wientjes, A. H. J. Kolk, B. G. Spratt, and N. Nanninga, J. Bacteriol. 171:1393-1401). They could be specified by studying monoclonal antibody binding patterns to amino- and carboxy-terminal truncated PBP 1B molecules. Monoclonal antibodies against conformational epitopes, with the exception of one epitope area, did not recognize PBP 1B molecules that had not been translocated across the membrane. Apparently, translocation is required for PBP 1B to fully obtain its native conformation.