A 1994 to 1995 national Haemophilus influenzae surveillance study of 1910 strains showed that 13 strains (0.7%) were resistant to amoxicillin/clavulanic acid (MIC, > or = 8/4 micrograms/ml). These and other selected strains were investigated further in this study. Susceptibility of the surveillance study strains was determined with the commercial microdilution trays used in the original study and in triplicate with reference broth microdilution trays prepared by the investigators, as well as by Etest and disk diffusion. Amoxicillin/clavulanic acid resistance was confirmed for only one of the surveillance study strains. This strain produced double zones of growth with Etest and disk-diffusion methods, with the double zone containing spheroplasts. When the amoxicillin/clavulanic acid MICs of all beta-lactamase positive strains were compared, MIC results obtained with surveillance study trays and the Etest were one to two dilutions higher than MICs obtained with reference trays. The distribution and modal amoxicillin/clavulanic acid MICs of beta-lactamase-positive and -negative strains was essentially the same for a comparison group of strains using reference trays, in contrast to a fourfold higher modal MIC for beta-lactamase-positive strains using surveillance study reagents and strains. These differences in MICs could be attributed to variations in inoculum, the presence of spheroplasts, and/or a difference in potency of amoxicillin and/or clavulanic acid in the tray and Etest reagents used. Methods for assessing the adequacy of the clavulanic acid content are not adequate, amoxicillin control values and a beta-lactamase-positive H. influenzae control strain are required.