Penicillin-binding protein (PBP) patterns of penicillin-resistant laboratory-constructed transformants were compared with the PBP profiles of 26 clinical isolates of Streptococcus pneumoniae. For transformation studies DNA from a penicillin-resistant clinical isolate was used to transform a susceptible laboratory strain. Penicillin resistance was achieved in two transformation cycles. The frequency of transformation appeared to be dependent on the genetic status of the recipient used for the second transformation cross. Penicillin resistance was also attained in a single transformation round when time was allowed for full expression of random multiple transformations. PBP 2b was the first PBP to show an alteration in penicillin-binding affinity. This PBP was not easily detected in those transformants for which penicillin MICs exceeded 0.2 mg/l. The PBP profiles of the clinical isolates were complex. In addition to previously-described PBPs, new intermediate classes were demonstrated. No correlation between PBP profile and susceptibility was observed with clinical isolates except that PBP-2b exhibited molecular weight changes in moderately susceptible strains.