Microdilution susceptibility tests are being performed routinely and in increasing numbers in many clinical laboratories. Microtiter plates for these assays are usually prepared in large batches and stored frozen until needed. Studies were performed to investigate the stability at -20C and at -70C of ampicillin, carbenicillin, ticarcillin, gentamicin, tobramycin, amikacin, chloramphenicol, and cephalothin, using Enterobacter aerogenes, Proteus mirabilis, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella as test organisms. The stability at -20C and -70C of clindamycin, vancomycin, cephalothin, cefazolin, oxacillin, methicillin, nafcillin, and penicillin was analyzed using Staphylococcus eureus, Staphylococcus epidermidis, Enterococcus, and alpha Streptococcus as the test organisms. No significant deterioration of any of the antibiotics was noted after ten weeks of storage at -70C, as detected by minimum inhibitory concentration determinations. No significant differences were noted at -20C for clindamycin, chloramphenicol, vancomycin, cefazolin, cephalothin, gentamicin, tobramycin, and amikacin. By ten weeks' storage at -20C, significant deterioration of carbenicillin, ticarcillin, ampicillin, penicillin, methicillin, oxacillin, and nafcillin was observed.