BACKGROUND: Bupivacaine, clindamycin, and gentamicin inhibit neuromuscular (NM) conduction. When they are combined, they may synergistically reduce the effective concentration of each to the therapeutic concentration in augmenting rocuronium-induced NM block. Thus, the aim of this study was to investigate whether combinations of the three drugs, at around their therapeutic concentrations, potentiate rocuronium-induced NM block. METHODS: Fifty-seven left-phrenic nerve hemidiaphragms (Male S-D rats, 150-250 g) were hung in a 20-ml organ bath filled with Krebs solution. Three consecutive single-twitch tensions (0.1 Hz) and one tetanic tension (50 Hz for 1.9 s) were obtained. A Krebs solution was premixed with concentration sets of bupivacaine and clindamycin, bupivacaine and gentamicin, or bupivacaine, clindamycin and gentamicin. Then, the concentration of rocuronium was cumulatively increased in the Krebs solution (1, 3, 5, 7, 9, 12, 14, 16, 18, and 20 microM) until an 80% to 90% reduction in single twitch was attained. The effective concentrations for each experiment were determined with the probit model. RESULTS: The combinations of bupivacaine, clindamycin, and gentamicin enhanced rocuronium-induced NM block. When the three drugs were applied simultaneously, their concentrations were reduced to near-therapeutic levels in potentiating the action of rocuronium. CONCLUSIONS: Bupivacaine, clindamycin, and gentamicin blocked NM conduction, and when all three drugs were applied together, they augmented rocuronium-induced NM block at their near-therapeutic concentrations. Clinicians should be aware of the cooperability in NM block between drugs that interrupt NM conduction.