Levobupivacaine is the isolated S(-)-stereoisomer of racemic bupivacaine. Important pharmacodynamic properties of levobupivacaine have not been determined for the femoral three-in-one block. In this randomized, controlled, double-blinded trial, we studied 60 ASA physical status I-III patients scheduled for surgery of the lower limb. A nerve-stimulator-guided three-in-one block was performed as supplemental analgesic therapy with 20 mL of bupivacaine 0.5% (n = 20), levobupivacaine 0.5% (n = 20), or levobupivacaine 0.25% (n = 20). Sensory onset time, quality of blockade, and duration of blockade were assessed by pinprick test in the central sensory innervation region of the femoral nerve (distribution of the anterior femoral cutaneous nerve). A rating scale from 100% (normal sensation) to 0% (no sensation at all) as compared with the contralateral leg was used. No significant difference in sensory onset time among the three local anesthetic solutions was observed (mean [95% confidence interval]): bupivacaine 0.5%, 27 min (20-33 min); levobupivacaine 0.5%, 24 min (18-30 min); and levobupivacaine 0.25%, 30 min (23-36 min) (P = 0.49). The analgesic quality of the blockade was also not significantly different among the three groups, whereas a complete sensory block was achieved in significantly fewer patients in the levobupivacaine 0.25% group (P = 0.02). The duration of blockade was significantly shorter with levobupivacaine 0.25% compared with the other groups: bupivacaine 0.5%, 1053 min (802-1304 min); levobupivacaine 0.5%, 1001 min (844-1158 min); and levobupivacaine 0.25%, 707 min (551-863 min) (P = 0.01). Levobupivacaine 0.5% is recommended instead of bupivacaine 0.5% for the three-in-one block.