We conducted a retrospective study to update the bacteriology of 46 cases of anaerobic empyema that were originally studied between 1976 and 1993 at the Wadsworth Anaerobic Bacteriology Clinical Research Laboratory (Los Angeles). Anaerobic bacteriologic studies were completed for all 46 pleural fluid specimens, and aerobic bacteriologic studies were completed for 41 of these specimens. Thirty-seven clinical charts were available for review. A total of 161 anaerobic isolates (3.5 per patient) representing 64 species or groups were recovered. The most common isolates were as follows: Fusobacterium nucleatum (19); Prevotella oris-buccae group (13, 9 of which were P. oris); Bacteroides fragilis group (11, 4 of which were B. fragilis); pigmented Prevotella species (17, 8 of which were in the Prevotella intermedia-nigrescens group); Peptostreptococcus species (17, 9 of which were Peptostreptococcus micros); Eubacterium species (7); Lactobacillus species (8); Actinomyces species (7); and Clostridium species (7). Nineteen if the cases were of purely anaerobic etiology; of these, eight were caused by a single organism: F. nucleatum (five cases); B. fragilis (two cases); and Prevotella mangus (one case). Of the 45 aerobic isolates (1.1 per patient), viridans streptococci were most common (21 isolates), followed by group D nonenterococcal streptococcus (four isolates). Only nine gram-negative rods (six enteric and three nonenteric organisms) and one Staphylococcus aureus isolate were recovered. The susceptibility to penicillin of 64 isolates was examined with the use of the spiral gradient method; 21 (33%) of these isolates were beta-lactamase positive (MICs ranged from 1.1 to > or = 54 micrograms/mL vs < or = 0.27 micrograms/mL for beta-lactamase-negative strains).