The penicillin-binding proteins (PBPs) of five penicillin tolerant group A streptococci and their isogenic non-tolerant strains, and seven unrelated non-tolerant group A streptococci were compared. PBPs from late logarithmic cultures were labelled in vitro with 3H-benzylpenicillin and analysed by SDS-PAGE and fluorography. The PBP patterns for all non-tolerant strains were identical. This pattern differed markedly from that for penicillin tolerant strains, both qualitatively and quantitatively. The most striking change in penicillin tolerant strains was decreased binding of 3H-penicillin to PBP 3 and increased binding to PBP 5, while PBP 2a was replaced by a new PBP (PBP 2a') of lower electrophoretic mobility. Tolerance was lost during storage but could be restored by consecutive transfers on to penicillin gradient agar plates. At the same time the PBP profiles of these strains became identical to those found for stable tolerant strains. These results suggest the possibility that PBP 2a' and PBP 5 in combination with other PBP alterations play a role in penicillin tolerance found in group A streptococci.