A quantitative description of ionophore-mediated ion transport is important in understanding ionophore activity in biological systems and developing ionophore applications. Herein, we describe the direct measurement of the electrical current resulting from K + transport mediated by individual valinomycin (val) ionophores.Step fluctuations in current measured across a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer suspended over a ∼400 nm radius glass nanopore result from dynamic partitioning of val between the bilayer and torus region, effectively increasing or decreasing the total number of val present in the membrane. In our studies, approximately 30 val are present in the membrane on average with a val entering or leaving the bilayer approximately every 50 s, allowing measurement of changes in electrical current associated with individual val. The single-molecule val(K + ) transport current at 0.1 V applied potential is (1.3 ± 0.6) × 10 −15 A, consistent with estimates of the transport kinetics based on large val ensembles. This methodology for analyzing single ionophore transport is general and can be applied to other carrier-type ionophores.