Recent isolations of strains of Haemophilus influenzae resistant to ampicillin necessitate the development of a rapid, dependable, reproducible method of determining their antibiotic susceptibility. An agar-dilution method permitting susceptibility determinations on clinical specimens within 6-18 hours of specimen collection was designed. Chocolate agar biplates were made with one side having no additive and the other containing 2 mug/ml ampicillin. Seventy clinical specimens (cerebrospinal fluid, joint fluid, ear fluid, pleural fluid, blood culture broth) were streaked directly onto both sides of the plates when received in the laboratory and incubated at 35-37 C in 10% CO2. Reliable, readable results were usually available within 6-18 hours of receipt of the specimen and correlated completely with minimum inhibitory concentrations (MIC) determined by the agar-dilution plate method, although standard disk susceptibilities occasionally indicated false resistance. Susceptible strains (MIC less than 2 mug/ml) grew on the antibiotic-free side of the biplate only. The rapid determination of ampicillin susceptibility allows optimal antibiotic selection for the treatment of Haemophilus influenzae infections with early discontinuation of potentially toxic supplementary drugs.