A total of 12 non-epidemiologically related clinical isolates of Streptococcus mitis that showed different levels of resistance to penicillin were studied. Membrane-protein profiles and penicillin-binding protein (PBP) patterns showed a great polymorphism; and patterns of 4-7 PBPs, with sizes that ranged from approximately 101 kDa to approximately 40 kDa, were detected in each strain. No association could be found between PBP pattern and resistance level to penicillin among these isolates. Arbitrarily primed PCR confirmed the genetic diversity among this group of streptococci. One of the isolates of intermediate level of resistance to penicillin, which showed a PBP pattern similar to that of the high-resistance strains, was used as a laboratory model to analyse the mechanism underlying high-resistance acquisition by these strains. A 14-fold increase in penicillin resistance was obtained after a single selection step, which resulted in a decrease in penicillin affinity for PBP1. The size of this PBP (92 kDa) and the differences in PBP profiles of the penicillin-resistant clinical isolates suggest the existence in S. mitis of PBP-mediated mechanisms to acquire high-level resistance to penicillin, among which alterations in PBP1 seem to play a main role, in contrast to the PBP2X mediated mechanism described for other streptococci.