The dye ruthenium red (RuR) has diverse experimental uses, including block of ion channels. RuR is a well described antagonist of one class of intracellular Ca2+ release channels, the ryanodine receptors, but recently this compound has also been identified as a putative blocker of voltage-gated calcium channels of the surface membrane involved in neurotransmitter release. Using electrophysiological methods, we have studied the action of RuR upon pure populations of neuronal voltage-gated ion channels heterologously expressed in Xenopus laevis oocytes. All four channel types studied, including class A (P/Q-type), class B (N-type), class C (L-type), and class E channels, are sensitive to RuR, with IC50 values ranging from 0.7 to 67.1 microM. Block of class C and class E channels most likely results from 1:1 binding of ruthenium red at a site in the extracellular entrance to the pore, resulting in obstruction of permeant ion flux through these channels. The mechanism of block of class A and class B channels is more complex, requiring binding of more than one molecule of RuR per channel.