At the leaf scale, it is a long-held assumption that stomata close at night in the absence of light, causing transpiration to decrease to zero. Energy balance models and evapotranspiration equations often rely on net radiation as an upper bound, and some models reduce evapotranspiration to zero at night when there is no solar radiation. Emerging research is showing, however, that transpiration can occur throughout the night in a variety of vegetation types and biomes. At the ecosystem scale, eddy covariance measurements have provided extensive data on latent heat flux for a multitude of ecosystem types globally. Nighttime eddy covariance measurements, however, are generally unreliable because of low turbulence. If significant nighttime water loss occurs, eddy flux towers may be missing key information on latent heat flux. We installed and measured rates of sap flow by the heat ratio method (Burgess et al. 2001) at two AmeriFlux (part of FLUXNET) sites in California. The heat ratio method allows measurement and quantification of low rates of sap flow, including negative rates (i.e., hydraulic lift). We measured sap flow in five Pinus ponderosa Dougl. ex Laws. trees and three Arctostaphylos manzanita Parry and two Ceanothus cordulatus A. Kellog shrubs in the Sierra Nevada Mountains, and in five Quercus douglasii Hook and Arn. trees at an oak savanna in the Central Valley of California. Nocturnal sap flow was observed in all species, and significant nighttime water loss was observed in both species of trees. Vapor pressure deficit and air temperature were both well correlated with nighttime transpiration; the influence of wind speed on nighttime transpiration was insignificant at both sites. We distinguished between storage-tissue refilling and water loss based on data from Year 2005, and calculated the percentage by which nighttime transpiration was underestimated by eddy covariance measurements at both sites.