The pathway(s) for copper (Cu) uptake across the mucosal membrane into intestinal cells has not been elucidated, and may include Cu uptake by the Cu-specific pathway encoded by Ctr1 or via Cu uptake through epithelial sodium channels (ENaCs). Our laboratory has developed a model of intestinal Cu absorption using a comparative physiological and genomic approach (Handy et al. 2000; Handy et al. 2002). In this study we aimed to explore whether or not Cu entry into intestinal cells was Na⁺-sensitive. Copper accumulation in freshly isolated intestinal cells from rainbow trout was measured by ICP-AES after exposure to 0-800 μM CuSO4 for 15 minutes. Cu accumulation by cells was 1.88 ± 0.52 compared with 0.05 ± 0.01 nmol Cu/mg cell protein/h with external Cu (Cu_o) of 800 μM and no-added Cu_o respectively (mean ± S.E.M., n = 6; Kruskal Wallis, P<0.05). Deduction of a rapid Cu accumulation on/in cells at time zero (about 12% of the total Cu when Cu_o was 800 μM) revealed a saturable uptake curve which reached a plateau at 400 μM Cu_o (Km, 216 μM Cu_o; Vmax, 1.09 nmol Cu/mg cell protein/h, 140 mM NaCl throughout). Incubating cells at 4^oC did not stop Cu accumulation. Lowering external Na⁺ to 11 mM (low Na_+o) generally decreased Cu accumulation into the cells over 15 min. In low Na⁺_o conditions Cu accumulation was exponential (non-saturable). Na⁺-insensitive Cu accumulation dominated (59% of total Cu accumulation) when Cu_o was 400 μM or less. At high Cu_o (800 μM), removal of Na⁺ caused a 45% increase in Cu accumulation. Pre incubation of cells with epithelial Na⁺ channel (ENaC) blocking agents for 15 minutes (normal NaCl throughout) caused Cu accumulation to increase by 40 fold (100 μM phenamil), 21 fold (10 μM CDPC) or 12 fold (2 mM amiloride) when Cu_o was 800 μM compared to drug-free controls (all significantly different from drug-free controls, Kruskal Wallis, P<0.05). Lowering external chloride (Cl^–_o) from 131.6 to 6.6 mM (replaced by Na⁺ gluconate) caused Cu accumulation to increase 11 fold when Cu_o was 800 μM. Application of 0.1 mM DIDS (normal Cl^–_o) caused a similar effect. Lowering external pH from 7.4 to pH 5.5 produced a 17 fold, saturable, increase in Cu accumulation which was not explained by increased instantaneous Cu accumulation on/in cells at low pH. We conclude that Cu accumulation by intestinal cells is mainly Na⁺-insensitive and more characteristic of a Ctr1-like pathway than Cu uptake through ENaCs.