Marquez, DiegoStuart-Williams, HilaryFarquhar, GrahamBusch, Florian A.2023-04-110028-646Xhttp://hdl.handle.net/1885/288668Cuticular conductance to water (gcw) is difficult to quantify for stomatous surfaces due to the complexity of separating cuticular and stomatal transpiration, and additional complications arise for determining adaxial and abaxial gcw. This has led to the neglect of gcw as a separate parameter in most common gas exchange measurements. Here, we describe a simple technique to simultaneously estimate adaxial and abaxial values of gcw, tested in two amphistomatous plant species. What we term the ‘Red-Light method’ is used to estimate gcw from gas exchange measurements and a known CO2 concentration inside the leaf during photosynthetic induction under red light. We provide an easy-to-use web application to assist with the calculation of gcw. While adaxial and abaxial gcw varies significantly between leaves of the same species we found that the ratio of adaxial/abaxial gcw (γn) is stable within a plant species. This has implications for use of generic values of gcw when analysing gas exchange data. The Red-Light method can be used to estimate total cuticular conductance (gcw-T) accurately with the most common setup of gas exchange instruments, i.e. a chamber mixing the adaxial and abaxial gases, allowing for a wide application of this technique.The authors thank CONICYT, Doctorado Becas Chile/2015Folio: 72160160 and the ARC Centre of Excellence for Transla-tional Photosynthesis for funding part of the research; Suan ChinWong for providing technical support; the Centre for AdvancedMicroscopy at the Australian National University (ANU) forinstruments and technical assistance; ANU Plant Services for taking care of the plant material.application/pdfen-AU© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundationhttps://creativecommons.org/licenses/by/4.0/202210.1111/nph.175882022-01-23Creative Commons Attribution License