1. The quaternary derivative of the potent verapamil-analogue, (-)-D888, (qD888, 4-cyano-4-(3,4-dimethoxyphenyl)-N-[2-(3-methoxy phenyl)ethyl]-N,N,5-trimethyl-1-hexanaminium) was synthesized as a novel membrane-impermeable probe to study the localization of phenylalkylamine (PAA) effector domains on L-type Ca2+ channels. Channel block by qD888 was investigated in smooth muscle-like (A7r5) cells after extra- and intracellular application by use of the whole-cell configuration of the patch clamp technique. 2. Extracellularly applied qD888 inhibited Sr2+ (Isr) (IC50 = 90 microM) and Na+ (IC50 = 27 microM) inward currents through L-type Ca(2+)-channels mainly in a resting-state-dependent manner. Structurally closely related quaternary PAAs (e.g. D890) were ineffective after extracellular application. 3. QD888 also blocked Isr from the cytoplasmic side, as did other quaternary PAAs (D890, D575). Intracellular block was clearly dependent on channel opening, which resulted in pronounced use-dependence. 4. We conclude that qD888 blocks L-type Ca2+ channels not only from the intracellular side, via interaction with the classical PAA binding domain, but also from the extracellular channel surface. The properties of Ca2+ channel block together with previous biochemical and structural data suggest that extracellular block may be mediated by a site that also confers tonic block by quaternary benzothiazepines.