The objective of the present study was to employ an enriched stable-isotope approach to characterize Zn uptake in the gills of rainbow trout (Oncorhynchus mykiss) during acute Zn exposures in hard water (???140 mg/L as CaCO ₃) and soft water (???30 mg/L as CaCO₃). Juvenile rainbow trout were acclimated to the test hardnesses and then exposed for up to 72 h in static exposures to a range of Zn concentrations in hard water (0-1,000 ??g/L) and soft water (0-250 ??g/L). To facilitate detection of new gill Zn from endogenous gill Zn, the exposure media was significantly enriched with ⁶⁷Zn stable isotope (89.60% vs 4.1% natural abundance). Additionally, acute Zn toxicity thresholds (96-h median lethal concentration [LC50]) were determined experimentally through traditional, flow-through toxicity tests in hard water (580 ??g/L) and soft water (110 ??g/L). Following short-term (???3 h) exposures, significant differences in gill accumulation of Zn between hard and soft water treatments were observed at the three common concentrations (75, 150, and 250 ??g/L), with soft water gills accumulating more Zn than hard water gills. Short-term gill Zn accumulation at hard and soft water LC50s (45-min median lethal accumulation) was similar (0.27 and 0.20 ??g/g wet wt, respectively). Finally, comparison of experimental gill Zn accumulation, with accumulation predicted by the biotic ligand model, demonstrated that model output reflected short-term (<1 h) experimental gill Zn accumulation and predicted observed differences in accumulation between hard and soft water rainbow trout gills. Our results indicate that measurable differences exist in short-term gill Zn accumulation following acclimation and exposure in different water hardnesses and that short-term Zn accumulation appears to be predictive of Zn acute toxicity thresholds (96-h LC50s). ?? 2009 SETAC.