A novel approach for setting interpretive breakpoints in disk diffusion antibiotic susceptibility testing according to determined minimum inhibitory concentration (MIC) limits is described, using the method of single-strain regression analysis. The procedure was tested on reference strains Staphylococcus aureus (ATCC 25923), Streptococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853), using published results from cefoperazone disk diffusion experiments. The correlation between logarithm of the disk content and inhibition zone diameter squared was linear, excluding three endpoint values. When constants A and B in the new regression line equation were calculated for the four strains, all four showed different regression lines. Zone diameters corresponding to various MICs were calculated for a disk content of 75 micrograms. The values obtained for the four strains were 20.1, 20.9, 24.9, and 25.8 mm, respectively, for an MIC of 16 micrograms/ml, and 15.7, 15.7, 22.3, and 17.9 mm, respectively, for an MIC of 64 micrograms/ml. The following zone diameter breakpoints were determined for the "I" (intermediate) category, using a 75-micrograms disk: S. aureus, 18 to 15 mm; S. faecalis, 23 to 13 mm; E. coli, 20 to 17 mm; and P. aeruginosa, 20 to 17 mm.