Voltage-gated ion channels are integral membrane proteins that conduct ions selectively and are regulated by drugs binding to specific sites, but available biochemical techniques have only broadly defined these sites. For the L-type calcium channel's 1,4-dihydropyridine (1,4-DHP) binding site—whose precise location remains unknown—we designed a homologous series of permanently charged 1,4-DHP probes: a 1,4-DHP pharmacophore linked to a quaternary ammonium group via a polymethylene chain of varying length. The charged group anchors the molecule at the extracellular membrane surface, restricting access of the 1,4-DHP moiety to the intramembrane binding site based on chain length; binding affinity thus depends on this access. We synthesized these probes (via Hantzsch condensation and quaternization) and tested their inhibition of [³H]-(+)-PN200-110 binding in rat ventricular myocytes. Binding affinity optimized at a minimal chain length of n=8 (compound 3c), with no significant improvements for longer chains (n=10, 12). Molecular modeling showed the distance from the charged group's quaternary nitrogen to the 1,4-DHP ester oxygen is ~11 Å. These results indicate the 1,4-DHP binding site is in the extracellular face of the membrane bilayer, at a depth of ~11 Å from the membrane surface. This novel series of permanently charged probes provides a new approach to localize intramembrane drug binding sites on ion channels, with potential utility for other voltage-gated ion channels, G-protein-coupled receptors, and ligand-gated ion channels.