The penicillin MIC of 2 Streptococcus pneumoniae clinical isolates was increased 100-fold (from 0.02 to 2.0 micromilligrams) and 20-fold (from 0.5 to 10.0 micromilligrams) through gradual exposure of the bacteria to increasing concentrations of penicillin in the laboratory. In both mutants the affinity of all four high molecular mass penicillin binding proteins (PBPs) for penicillin was drastically reduced accompanied by major changes in the composition of peptidoglycan as resolved by HPLC. The ratio of crosslinked to monomeric peptides became virtually inverted in the resistant cell walls with monomers representing two-thirds of the muropeptide species. The proportion of the crosslinked tri-tetra dimer, a major component of the cell wall of the original isolates, decreased to one-third or one-sixth of its normal representation, while the amounts of tripeptide monomers with an alanyl-serine substitution on the lysine epsilon amino group increased by close to a factor of two. The growth rates of both mutants decreased by a factor of approximately two, as compared to the original bacteria.