A clone of Hungarian pneumococcal strains has recently been isolated with notably high levels of beta-lactam resistance (penicillin MIC, 16 microg/mL; cefotaxime MIC, 4 microg/mL). The role that each penicillin-binding protein (PBP) plays in the development of resistance in these strains was investigated via transformation of susceptible strain R6 with pbp DNA from resistant strain 3191. Transformation of strain R6 with pbp2X DNA resulted in transformants with penicillin and cefotaxime MICs of 0.06 and 0.25 microg/mL, respectively. Further introduction of pbp2B and 1A DNA increased penicillin MICs to 0.25 and 4 microg/mL, respectively. Transformation of strain R6 with a combination of pbp2X and pbp1A DNA produced R63191/2X/1A strains with an unexpected low cefotaxime MIC of 0.5 microg/mL. This low-level of cefotaxime resistance was surprisingly increased from 0.5 to 2 microg/mL in R63191/2X/2B/1A strains. This suggests the involvement of altered PBP 2B in cefotaxime resistance. Therefore, within this particular setting of resistance, the environmental presence of selectors for altered PBP 2B (penicillin or piperacillin) are required for the development of high-level cefotaxime resistance. The MICs of R63191/X/2B/1A strains never reached the MICs of the donor strain. Full MICs of the donor were eventually reached by transforming R63191/2X/2B with chromosomal3191 DNA. Resultant transformants revealed the introduction of altered PBP 1A, while unaltered PBPs 1B, 2A, and 3 proved that these PBPs were not involved in resistance. A non-PBP resistance determinant has therefore made up the difference in resistance between R63191/2X/2B/1A and donor strain 3191. Beta-lactamase activity and efflux systems have so far been eliminated as causes of resistance. This resistance determinant represents a novel mechanism for beta-lactam resistance in clinical isolates of pneumococci, operates at the highest level of resistance, and remains to be identified.