Xylem sap pH (pH(X)) is critical in determining the quantity of inorganic carbon dissolved in xylem solution from gaseous [CO2] measurements. Studies of internal carbon transport have generally assumed that pH(X) derived from stems and twigs is similar and that pH(X) remains constant through time; however, no empirical studies have investigated these assumptions. If any of these assumptions are violated, potentially large errors can be introduced into calculations of dissolved CO2 in xylem and resulting estimates of internal carbon transport. We tested the validity of assumptions related to pH(X) in Populus deltoides L. with a series of non-manipulative experiments. The pH(X) derived from stems and twigs was generally similar and remained relatively constant through a diel period. The only exception was that pH(X) derived from lower stem sections at night was higher than that derived from twigs. The pH x derived from stems was similar on clear days when solar radiation and vapor pressure deficit (VPD) were similar, but higher on an overcast day when solar radiation and VPD were lower. Similarly, cloudy conditions immediately before an afternoon thunderstorm increased pH(X) derived from twigs. The pH(X) derived from twigs remained similar when measured on sunny afternoons between July and October. Our results suggest that common assumptions of pH(X) used in studies of internal carbon transport appear valid for P. deltoides and further suggest pH(X) is influenced by environmental factors, such as solar radiation and VPD that affect transpiration rates.