Documented ampicillin treatment failures of systemic Haemophilus influenzae type b infections have been associated with synthesis of a TEM-1 beta-lactamase. A patient with H. influenzae type b meningitis in whom ampicillin treatment failed is described; the isolate was beta-lactamase-negative according to the cell suspension chromogenic cephalosporin assay. The false-negative result occurred in a strain which elaborated a novel, plasmid-mediated beta-lactamase with characteristics which distinguish it from TEM-1 beta-lactamase. Clinically important ampicillin resistance in H. influenzae type b occurs by mechanisms other than by synthesis of TEM-1 beta-lactamase. Diagnostic microbiology laboratories should perform antibiotic susceptibility tests in addition to tests for beta-lactamase production.