The penicillin-binding protein (PBP) profiles of penicillin-susceptible and -resistant clinical isolates of Streptococcus mitis varied even with strains with similar minimal inhibitory concentrations (MICs). S. mitis NCTC 10712 was used as a DNA recipient to investigate PBP alterations which could occur as a result of spontaneous mutation and intra- and interspecific transfer of penicillin resistance genes. S. mitis NCTC 10712 possesses seven major PBPs ranging in molecular mass from 49-82 kDa. Two S. mitis and two Streptococcus pneumoniae penicillin-resistant clinical isolates were used as donors in transformation experiments with S. mitis NCTC 10712 (MIC 0.03-mu-g/ml) as the recipient. Transformants with MICs greater than 1-mu-g/ml were obtained with both S. mitis and S. pneumoniae donor DNA. Depending on the source of the donor DNA and level of resistance achieved, transformants showed reduced penicillin-binding affinities of PBPs 2, 3, 4, 5, and 6. The most consistent PBP alteration associated with increasing resistance in S. mitis NCTC 10712 was seen with PBP 3 (74 kDa).