Penicillin-resistant oral streptococci constitute the genetic reservoir for beta-lactam resistance in S. pneumoniae. Here we report the isolation of clinical strains of S. mitis with unusually high MIC values for beta-lactam antibiotics; resistance to benzylpenicillin was 64 microg/ml and to cefotaxime 128 microg/ml. Among the beta-lactam compounds tested, only the carbapenems imipenem and meropenem showed MICs below 32 microg/ml. Both S. mitis strains were resistant to tetracycline and were highly resistant to aminoglycosides. Pulse field mapping of chromosomal DNA revealed identical patterns in both strains, indicating clonal identity of the two isolates. Using chromosomal S. mitis DNA, the laboratory strain S. pneumoniae R6 could be transformed in four successive steps to cefotaxime and benzylpenicillin resistance of 64 microg/ml. The results exemplify the importance of commensal streptococci for the development of cefotaxime resistance in S. pneumoniae.