Interstitial K⁺ concentrations were measured during one-legged knee-extensor exercise by use of microdialysis with probes inserted in the vastus lateralis muscle of the subjects. K⁺ in the dialysate was measured either by flame photometry or a K⁺-sensitive electrode placed in the perfusion outlet. The correction for fractional K⁺ recovery was based on the assumption of identical fractional thallium loss. The interstitial K⁺ was 4.19 ± 0.09 mM at rest and increased to 6.17 ± 0.19, 7.48 ± 1.18, and 9.04 ± 0.74 mM at 10, 30, and 50 W exercise, respectively. The individual probes demonstrated large variations in interstitial K⁺, and values >10 mM were obtained. The observed interstitial K⁺ was markedly higher than previously found for venous K⁺ concentrations at similar work intensities. The present data support a potential role for interstitial K⁺ in regulation of blood flow and development of fatigue.