Abstract. Studies of the isoprene emission rate in response to changes in photon-flux density and CO2 partial pressure were conducted using a recently developed on-line isoprene analyser combined with a gas exchange system and chlorophyll fluorometer. Upon darkening, the isoprene emission rate from leaves of aspen (Populus tremuloides Michaux.) began to decline immediately, demonstrating that the internal pool of isoprene, or its precursors, is small and that the instantaneous emission rate is tightly coupled to the rate of synthesis. A post-illumination burst of isoprene was observed within 5 min after darkening and lasted for 15–20 min in four isoprene-emitting species that were examined. In leaves of eucalyptus (Eucalyptus globulus Labill.), the magnitude of the post-illumination burst was dependent on the photon-flux density that existed before darkening, but not on ambient CO2 partial pressure. The dependence of the post-illumination burst on photon-flux density paralleled that for the steady-state rate of isoprene emission. A step-wise increase in intercellular CO2 partial pressure from 24.5 to 60 Pa resulted in an immediate decrease in isoprene emission rate and non-photochemical fluorescence quenching, but an increase in CO2 assimilation rate. Given the several recent studies that link isoprene emission to chloroplastic processes, the results of this study indicate that the linkage is not dependent on the rate of CO2 flux through the reductive pentose phosphate pathway, but rather on more complex relationships involving metabolites not appreciably influenced by CO2 partial pressure.