Background: In isolated nerves, tetrodotoxin (TTX) blocks nerve conduction longer than bupivacaine. In vivo, however, both substances block nerve conduction to an equal duration, presumably because the hydrophilic TTX binds only weakly to the perineural tissue. High molecular weight hyaluronic acid (HA) prolongs the action of local anaesthetics several-fold. We tested whether admixture of HA enhances the binding of TTX to the perineural tissue and thus induces an ultralong conduction block after a single application. Methods: In 12 anaesthetized rabbits, the minimal blocking concentrations of TTX, TTX and HA (TTX/HA) and bupivacaine with HA (bupivacaine/HA) were determined by blocking the natural spike activity of the aortic nerve. In 18 other animals, equipotent concentrations of either TTX, TTX/HA or TTX/bupivacaine/HA were applied topically to the aortic nerve. After disappearance of the spike activity, the wound was closed to simulate the clinical situation of a single shot nerve block. The time until recovery of spike activity was determined. The nerves were examined for signs of neurotoxicity 24 h after the application of the drugs. Data are presented as means ± SD and compared by ANOVA and Student's t-test for unpaired data. Results: The conduction block by TTX/bupivacaine/HA (10.1 ± 1.9 h) or TTX/HA (9.3 ± 1.0 h) was significantly longer than that of plain TTX (7.9 ± 1.0 h). Neurotoxicity was not observed. Conclusions: Both HA and HA/bupivacaine prolong the TTX-induced conduction blockade of the aortic nerve of rabbits in vivo. No signs of neurotoxicity were observed.