This paper describes a new approach to the calibration of thermal infrared measurements of leaf temperature for the estimation of stomatal conductance and illustrates its application to thermal imaging of plant leaves. The approach is based on a simple reformulation of the leaf energy balance equation that makes use of temperature measurements on reference surfaces of known conductance to water vapour. The use of reference surfaces is an alternative to the accurate measurement of all components of the leaf energy balance and is of potentially wide application in studies of stomatal behaviour. The resolution of the technique when applied to thermal images is evaluated and some results of using the approach in the laboratory for the study of stomatal behaviour in leaves of Phaseolus vulgaris L. are presented. Conductances calculated from infrared measurements were well correlated with estimates obtained using a diffusion porometer.