The first analysis of chloride fluxes and compartmentation in a non-excised plant system is presented, examining ten ecologically pertinent conditions. The short-lived radiotracer couple (38)Cl/(39)Cl was used as a Cl(-) tracer in intact barley ( Hordeum vulgare L. cv. Klondike) seedlings, which were cultured and investigated under four external [Cl(-)], from abundant (0.1 mM) to potentially toxic (100 mM). Chloride-nitrogen interactions were investigated by varying N source (NO(3)(-) or NH(4)(+)) and strength (0.1 or 10 mM), in order to examine, at the subcellular compartmentation level, the antagonism, previously documented at the influx level, between Cl(-) and NO(3)(-), and the potential role of Cl(-) as a counterion for NH(4)(+) under conditions in which cytosolic [NH(4)(+)] is excessive. Cytosolic [Cl(-)] increased with external [Cl(-)] from 6 mM to 360 mM. Cl(-) influx, fluxes to vacuole and shoot, and, in particular, efflux to the external medium, also increased along this gradient. Efflux reached 90% of influx at the highest external [Cl(-)]. Half-times of cytosolic Cl(-) exchange decreased between high-affinity and low-affinity influx conditions. The relationship between cytosolic [Cl(-)] and shoot flux indicated the presence of a saturable low-affinity transport system ("SLATS") responsible for xylem loading of Cl(-). N source strongly influenced Cl(-) flux to the vacuole, and moderately influenced Cl(-) influx and shoot flux, whereas efflux and half-time were insensitive to N source. Cytosolic pool sizes were not strongly or consistently influenced by N source, indicating the low potential for Cl(-) to act as a counterion to hyperaccumulating NH(4)(+). We discuss our results in relation to salinity responses in cereals.