The main mechanism of resistance of pneumococci to penicillin is the alteration of the affinities of penicillin binding proteins (PBPs) to --lactams by heterologous recombination events (4). For the present study, we have investigated the diversity of the penA (accession numbers AM779386 to AM779409), pbpX (AM779338 to AM779361), and pbp1A (AM779362 to AM779385) genes of 21 clinical Portuguese Streptococcus pneumoniae strains (randomly selected from the strain collection of the Antibiotic Resistance Unit at the National Institute of Health in Lisbon) in relation to their penicillin susceptibilities; four ATCC strains were also added to the sample. The analyzed sequences were compared to the R6 amino acid sequence. MICs to penicillin G were determined and interpreted as previously described (3). We found 21, 23, and 20 different alleles in PBP2B, PBP2X, and PBP1A, respectively; only 4, 5, and 8 alleles were already present in the DDBJ/EMBL/GenBank databases, respectively. In comparison to the amino acid sequence of strain R6, the average number of amino acid substitutions in strains susceptible (n 8), intermediate (n 10), and resistant (n 7) to penicillin were 3, 32, and 24 for PBP2B; 15, 52, and 23 for PBP2X; and 5, 46, and 28 for PBP1A, respectively. Among nonsusceptible strains, PBP2B presented short mosaics in Ala-Phe-Ser-Arg-Pro-Met (5/17), Ala-Phe-Ser-Val-Pro-Met (1/17), and Pro-Ala-Phe-Ser-Val-Pro-Thr (1/ 17) from residues 431 to 437; these mosaics were variants of those found by Dowson and others (4, 9).