The increasing number of penicillin-resistant clinical strains of Streptococcus pneumoniae has raised questions about the mechanism involved. We have isolated a large number of independent, spontaneous laboratory mutants with increasing resistance against either piperacillin or cefotaxime. Both classes of mutants showed a different pathway of penicillin-binding protein (PBP) alterations, and within each group of mutants the individual PBPs appeared to have changed at different resistance levels and in different sequences. The mutations led to decreased beta-lactam affinity and possibly to a reduction in the amount of protein present in the cell, but differences in apparent molecular weight, like those reported in low- and high-level resistant pathogenic strains, were not found. Some mutants showed a high degree of cross-resistance to a variety of penicillins and cephalosporins independently of the acquired PBP alterations, indicating that different genotypes can be responsible for the same phenotypic expression of resistance.