A series of isogenic pneumococcal transformants differing in their levels of penicillin resistance and containing altered penicillin-binding proteins were compared for their cell wall structures by using a recently developed technique that can resolve the peptidoglycan stem peptides of Pneumococcus strains to over 40 components (J. F. Garcia-Bustos, B. T. Chait, and A. Tomasz, J. Biol. Chem. 32:15400-15405). The stem peptides from the highly resistant transformants differed strikingly from those of the susceptible recipient strain, and the peptide patterns were almost identical to that of the DNA donor. Four peptides representing the major components in the walls of susceptible cells were replaced by six new peptides that were only minor components of susceptible cell walls. A remarkable common feature of these new species was their high alanine content. Amino acid analysis, sequencing, and mass spectrometry allowed the assignment of the extra alanine residues to dialanine or alanylserine cross bridges in the six new stem peptides. The common feature of the four peptide species that were present as major components in the susceptible walls, but became minor species in the resistant cells, was the absence of a cross bridge in at least one of the stem peptide components. We suggest that the extensive remodelling of cell wall synthetic enzymes that accompanies acquisition of penicillin resistance eventually also alters the reactivity of these proteins towards their natural substrates in cell wall synthesis. As a result, highly penicillin-resistant pneumococci will shift from the use of wall precursors with linear stem peptides to a preferential use of precursors containing the more-hydrophobic peptides carrying dialanyl or alanylserine cross bridges.