Amino acid residues F1760 and Y1767 in S6/Domain IV of voltage gated Na+ channels are part of the local anesthetic binding site in the inner mouth of the pore. Mutagenesis experiments have shown that use-dependent block (UDB) of the cardiac hNaV1.5 Na+ channel by the Class Ia anti-arrhythmic drug lidocaine is significantly reduced by alanine substitutions at these two sites. The antianginal drug ranolazine, which has structural similarity to lidocaine, was shown previously to block the cardiac late sodium current (INa) and to block peak and late INa in a use-dependent manner (Rajamani et al., 2009). As with lidocaine, UDB of peak INa by ranolazine was reduced in the F1760A mutant channel (Fredj et al., 2006; Wang et al., 2008). We used the F1760A and Y1767A mutants to investigate further the block by ranolazine of the closed, inactivated, and open states of the channel. To measure affinity to the open state, we used an inactivation-deficient hNaV1.5 channel (L409C/A410W, Edrich et al., 2005) in the presence and absence of the F1760A or Y1767A mutations. We found that both mutations (F1760A and Y1767A) significantly decreased UDB of peak INa, block of the open state, and block of late INa by ranolazine compared to WT channels (14-24 fold shift of the value of IC50 for ranolazine). The effect of the mutations on block by ranolazine of the resting and inactivated states was modest (1.3-1.9 fold shift of the value of IC50 for ranolazine). The results indicate that both the F1760A and Y1767A mutations reduce the effect of ranolazine to cause UDB and open state block of hNaV1.5.