We measured the DeltaPsi and DeltapH components of the transthylakoid proton motive force (pmf) in light-adapted, intact tobacco leaves in response to moderate heat. The DeltaPsi causes an electrochromic shift (ECS) in carotenoid absorbance spectra. The light-dark difference spectrum has a peak at 518 nm and the two components of the pmf were separated by following the ECS for 25 s after turning the light off. The ECS signal was deconvoluted by subtracting the effects of zeaxanthin formation (peak at 505 nm) and the qE-related absorbance changes (peak at 535 nm) from a signal measured at 520 nm. Heat reduced DeltapH while DeltaPsi slightly increased. Elevated temperature accelerated ECS decay kinetics likely reflecting heat-induced increases in proton conductance and ion movement. Energy-dependent quenching (qE) was reduced by heat. However, the reduction of qE was less than expected given the loss of DeltapH. Zeaxanthin did not increase with heat in light-adapted leaves but it was higher than would be predicted given the reduced DeltapH found at high temperature. The results indicate that moderate heat stress can have very large effects on thylakoid reactions.