Various amino acid residues within the inwardly rectifying potassium channel ROMK (Kir1.1) influence its expression at the plasma membrane (e.g. Lin et.al. 2002). However, domains that determine polarized expression of ROMK are so far unidentified. Here,we report the effects of C-terminal deletion of ROMK2 (Kir1.1b) on channel function and membrane targeting in Xenopus oocytes and MDCK cells. A C-terminus deletion mutation (A170X) was made by recombinant PCR. cDNAs encoding rat wild type ROMK2 (RK) tagged at the N-terminus with EGFP, and A170X were sub-cloned into Xenopus (pTLN) and fusion (pEGFP-C2) expression vectors. Female Xenopus laevis were killed humanely and stage V – VI oocytes isolated. Oocytes were injected with 50 nl of RNAse-free H₂O containing 0 or 5 ng (0.1 ng/nl^-1) cRNA encoding either EGFP, RK or A170X. Channel activity was assessed 3 – 4 days post injection at room temperature using two-electrode voltage clamp. The bath contained (in mM) NaCl 96; KCl 2; MgCl₂ 1; CaCl₂ 1.8; HEPES 5, (pH 7.5 ± 0.02) ± 5 mM BaCl.To determine the location of EGFP-tagged proteins, oocytes were fixed (1% paraformaldehyde) and sectioned (10 – 15 µm) using a cryostat at -25 ^oC. Type II MDCK cells were cultured under standard conditions on permeable filter inserts (Costar). Confluent monolayers were transfected using Lipofectamine 2000™ with 1 µg cDNA coding for A170X, RK or EGFP (n = 4 – 7), and were fixed after 48 hours with methanol:acetone (7:3 v/v). Localisation of fluorescence was determined by confocal laser scanning microscopy (λ_EX = 488 nm). Data are presented as mean ± S.E.M. and statistical significance was determined by one-way ANOVA and Tukey’s test. At a clamp potential of 0 mV, oocytes expressing RK displayed Ba²⁺ – sensitive outward currents of 3.36 ± 0.54 µA (n = 11). Fluorescence was observed at the plasma membrane. In contrast, oocytes expressing A170X exhibited Ba²⁺ – insensitive outward currents of 0.17 ± 0.05 µA (n = 9, P < 0.05); not significantly different from either H₂O injected (0.13 ± 0.01 µA, n = 8) or EGFP – expressing oocytes (0.14 ± 0.01 µA, n = 8). Oocytes expressing A170X or EGFP displayed uniform fluorescence throughout the cell but not at the plasma membrane (n = 3 – 5). In MDCK cells expressing EGFP, fluorescence was distributed throughout the cytoplasm but RK was targeted to the apical membrane. In contrast, A170X fluorescence was punctate and intracellular. These results underscore the importance of the C-terminus for membrane targeting of ROMK2. Further experiments are required to identify amino acid motifs that determine polarized sorting of these channel proteins.