A total of 175 isolates of anaerobic gram-negative bacilli were tested for beta-lactamase production by using a slide test modification of the chromogenic cephalosporin (Nitrocefin, Glaxo, Middlesex, England) assay and the iodometric slide test. Included isolates were Bacteroides melaninogenicus (46), B. fragilis (78), other Bacteroides isolates (21), Fusobacterium (25), and other gram-negative bacilli (5). Both slide tests detected 25 B. melaninogenicus isolates that were beta-lactamase producers (minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml). beta-Lactamase produced by the other gram-negative anaerobes could only be detected by the Nitrocefin assay. This assay was positive in 70 or 77 B. fragilis against which the minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml. Ten of 11 other species of Bacteroides against which the minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml were also Nitrocefin test positive. Minimal inhibitory concentrations of penicillin against all isolates of Fusobacterium and unidentifified gram-negative bacilli were less than or equal to 0.78 micrograms/ml and were Nitrocefin assay negative. beta-Lactamase-producing strains of B. melaninogenicus can be differentiated because both the slide iodometric and Nitrocefin assays will be positive, whereas beta-lactamase produced by other Bacteroides will only be detected by the Nitrocefin assay. Such penicillin-resistant isolates could be detected and reported to clinicians before final identification.