Background: Cardiotoxic effects of local anesthetics (LAs) involve inhibition of Na V1.5 voltagegated Na + channels. Metastatic breast and colon cancer cells also express Na V1.5, predominantly the neonatal splice variant (nNa V1.5) and their inhibition by LAs reduces invasion and migration. It may be advantageous to target cancer cells while sparing cardiac function through selective blockade of nNa V1.5 and/or by preferentially affecting inactivated Na V1.5, which predominate in cancer cells. We tested the hypotheses that lidocaine and levobupivacaine differentially affect (1) adult (aNa V1.5) and nNa V1.5 and (2) the resting and inactivated states of Na V1.5. Methods: The whole-cell voltage-clamp technique was used to evaluate the actions of lidocaine and levobupivacaine on recombinant Na V1.5 channels expressed in HEK-293 cells. Cells were transiently transfected with cDNAs encoding either aNa V1.5 or nNa V1.5. Voltage protocols were applied to determine depolarizing potentials that either activated or inactivated 50% of maximum conductance (V1/2 activation and V1/2 inactivation, respectively). Results: Lidocaine and levobupivacaine potently inhibited aNa V1.5 (IC 50 mean [SD]: 20  and 1 [0.6] μM, respectively) and nNa V1.5 (IC 50 mean [SD]: 17  and 3 [1.6] μM, respectively) at a holding potential of -80 mV. IC 50s differed significantly between lidocaine and levobupivacaine with no influence of splice variant. Levobupivacaine induced a statistically significant depolarizing shift in the V1/2 activation for aNa V1.5 (mean [SD] from -32 [4.6] mV to -26 [8.1] mV) but had no effect on the voltage dependence of activation of nNa V1.5. Lidocaine had no effect on V1/2 activation of either variant but caused a significantly greater depression of maximum current mediated by nNa V1.5 compared to aNa V1.5. Similar statistically significant shifts in the V1/2 inactivation (approximately -10 mV) occurred for both LAs and Na V1.5 variants. Levobupivacaine (1 μM) caused a significantly greater slowing of recovery from inactivation of both variants than did lidocaine (10 μM). Both LAs caused approximately 50% tonic inhibition of aNa V1.5 or nNa V1.5 when holding at -80 mV. Neither LA caused tonic block at a holding potential of either -90 or -120 mV, voltages at which there was little steady-state inactivation. Higher concentrations of either lidocaine (300 μM) or levobupivacaine (100 μM) caused significantly more tonic block at -120 mV. Conclusions: These data demonstrate that low concentrations of the LAs exhibit inactivation-dependent block of Na V1.5, which may provide a rationale for their use to safely inhibit migration and invasion by metastatic cancer cells without cardiotoxicity.