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 38C1/39C1 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 NH4+) 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 NO3-, and the potential role of Cl- as a counterion for NH4+ under conditions in which cytosolic [NH4+] 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 NH4+. We discuss our results in relation to salinity responses in cereals.