To quantify the relationship between temporal and spatial variation in tree transpiration, we measured sap flow in 129 trees with constant-heat sap flow sensors in a subalpine forest in southern Wyoming, USA. The forest stand was located along a soil water gradient from a stream side to near the top of a ridge. The stand was dominated by Pinus contorta Dougl. ex Loud. with Picea engelmannii Parry ex Engelm and Abies lasiocarpa (Hook.) Nutt. present near the stream and scattered individuals of Populus tremuloides Michx. throughout the stand. We used a cyclic sampling design that maximized spatial information with a minimum number of samples for semivariogram analyses. All species exhibited previously established responses to environmental variables in which the dominant driver was a saturating response to vapor pressure deficit (D). This response to D is predictable from tree hydraulic theory in which stomatal conductance declines as D increases to prevent excessive cavitation. The degree to which stomatal conductance declines with D is dependent on both species and individual tree physiology and increases the variability in transpiration as D increases. We quantified this variability spatially by calculating the spatial autocorrelation within 0.2-kPa D bins. Across 11 bins of D, spatial autocorrelation in individual tree transpiration was inversely correlated to D and dropped from 45 to 20 m. Spatial autocorrelation was much less for transpiration per unit leaf area and not significant for transpiration per unit sapwood area suggesting that spatial autocorrelation within a particular D bin could be explained by tree size. Future research should focus on the mechanisms underlying tree size spatial variability, and the potentially broad applicability of the inverse relationship between D and spatial autocorrelation in tree transpiration.