Here we present measurements of the stable isotope ratios of potassium (41K/39K) in three biological systems. We show that the ratio of 41K to 39K varies systematically: between the single-celled green alga Chlamydomonas reinhardtii and growth medium; between muscles of both euryhaline and stenohaline marine teleosts and seawater; and between blood plasma and red blood cells, muscles, cerebrospinal fluid, brain tissues, and urine in the terrestrial mammal Rattus norvegicus. Considered in the context of our current understanding of K+ transport in these biological systems, our results provide evidence that the fractionation of K isotopes depends on transport pathway and transmembrane transport machinery: K+ channels and paracellular transport through tight-junctions favor 39K whereas K+ pumps and co-transporters exhibit less isotopic fractionation. These results indicate that stable K isotopes can provide unique quantitative insights into the machinery and dynamics of K+ homeostasis in biological systems.