Osmoregulation in guard cells of intact, attached Vicia faba leaves grown under growth chamber and greenhouse conditions was studied over a daily light cycle of stomatal movements. Under both growth conditions guard cells had two distinct osmoregulatory phases. In the first (morning) phase, opening was correlated with K+ uptake and, to a lesser extent, sucrose accumulation. In the second (afternoon) phase, in which apertures were maximal, K+ content declined and sucrose became the dominant osmoticum. Reopening of the stomata after a CO2-induced closure was accompanied by accumulation of either K+ or sucrose, depending on the time of day, indicating that a single environmental signal may use multiple osmoregulatory pathways. Malate accumulation, correlated with K+ uptake, was detected under growth chamber but not greenhouse conditions, whereas Cl- was the main K+ counterion in the greenhouse. These results indicate that guard-cell osmoregulation in the intact leaf depends on at least two different osmoregulatory pathways, K+ transport and sucrose metabolism. Furthermore, the relative importance of the K+ counterions malate and Cl- appears to be environment-dependent.