A β-lactamase-producing strain of Clostridium clostridioforme isolated from human peritoneal fluid was examined by MIC testing and enzyme characterization. MICs of penicillins (64–512 mg/L) were higher than those of cephalosporins (8–128 mg/L); the strain was susceptible to cefoxitin (8 mg/L) and imipenem (1 mg/L). No enhancement of cephalosporin activity occurred when clavulanate was also added, but a limited degree of enhancement of penicillin activity (resulting in β-lactam MICs higher than available NCCLS breakpoints) occurred when clavulanate, sulbactam or tazobactam was added simultaneously. By contrast, addition of BRL 42715 with amoxycillin, ticarcillin or piperacillin led to a drop in β-lactam MICs from 512 to ≤ 1 mg/L, with a drop from 64 to 1 mg/L when BRL 42715 was added with cefotaxime. All inhibitors were added at fixed concentrations of 2 mg/L. As determined spectrophotometrically, the enzyme hydrolysed penicillin G, cloxacillin and piperacillin (Vmax values (%) 372, 1816, 1001, respectively relative to cephaloridine) more efficiently than cephalosporins (69–191, with cephaloridine as 100%). Km values (μM) varied between 30–308, μM (penicillins) and 2–20 μM (cephalosporins). Relative enzyme efficiency (relative VmaxKm, with cephaloridine as 100) varied from 21–100 (cephalosporins) and 8–77 (penicillins). ICS values (μM) with nitrocefin, piperacillin and penicillin G substrates (concentrations 20, 100 and 20 μM, respectively) were > 1000, 7, 3.5 (clavulanate); > 1000, 300, 59 (sulbactam), > 1000, 29, 7.7 (tazobactam); 0.0004, 0.001, 0.0018 (BRL 42715). The enzyme was not inhibited by EDTA, cefoxitin, cloxacillin or aztreonam, but was inhibited by pCMB. It was inducible by penicillin G and cefoxitin, and displayed a p1 of 4.2±0.1.