The presence and sequence variation of the murM gene were studied in a large collection (814 strains) of genetically diverse Streptococcus pneumoniae isolates, which included 27 different serogroups and both penicillin-resistant (423 isolates, 67 pulsed-field gel electrophoretic [PFGE] types) and intermediately penicillin-resistant (165 isolates, 66 PFGE types) and penicillin-susceptible (226 isolates, 135 PFGE types) strains. Diversity of the murM sequences was tested by hybridization with mainly two kinds of probes: one derived from the amplification of the nucleotide sequence between nucleotides 201 and 624 in the penicillin-susceptible laboratory strain R36A (murMA probe) and a second probe that amplified the comparable, highly divergent sequence in the penicillin-resistant strain Pen6 (murMB probe). The great majority of the strains (761 of 814), including both penicillin-susceptible and penicillin-resistant isolates, reacted exclusively with the murMA probe. A smaller group of penicillin-resistant strains (48 of 814 isolates) reacted only with the murMB DNA probe, and an additional 5 isolates reacted with both probes. High-pressure liquid chromatography analysis of the peptidoglycan of strains hybridizing with murMB showed that they invariably contained an increased proportion of branched peptides. Complete sequencing of murM from a group of penicillin-resistant isolates allowed the identification of a number of different murMB alleles that differed in the length and exact position of the divergent (Pen6 type) sequences within the particular murM. The close similarity of these divergent sequences in the various murM alleles suggests a possible common heterologous origin.