The Kir1.1 family of inwardly rectifying K⁺ channels (ROMK 1-3: Kir1.1 a-c) mediate K⁺ secretion across the apical membranes of the thick ascending limb and distal nephron (Hebert et al. 2005). However, the mechanisms that determine apical membrane targeting of Kir1.1 are not understood. N-Glycans determine surface expression of a number of membrane proteins (Muth & Caplan, 2003) and Kir1.1 contains a single glycosylation consensus sequence (NXS/T) at residues 98-100. In this study, we investigated the importance of glycosylation in membrane targeting of Kir1.1b. Filter-grown monolayers of MDCK type I cells stably expressing EGFP-Kir1.1b (WT) were treated overnight with cycloheximide (CX: 20 μg/ml) and allowed to recover for 3, 6 or 24 h either in the absence or presence of 10 µg/ml tunicamycin (TM) to inhibit glycosylation (n = 3-5 separate experiments). In a second series of experiments, monolayers of MDCK type II cells were transfected with cDNA coding for WT or a mutant (N98Q: made by QuickChange® mutagenesis) lacking the glycosylation site (n = 3-5 separate transfections). Intracellular localisation of EGFP-fusion proteins was determined by fluorescence confocal microscopy in conjunction with labelling of the apical membrane with TRITC conjugated wheat germ agglutinin (WGA) or peanut agglutinin (PNA) for MDCK I or II, respectively. In monolayers treated with CX for 17 h, WT levels were greatly reduced and fluorescence was not evident at the apical pole of the cells. When cells were washed and incubated in CX-free media for a further 3, 6 or 24 h, the levels of fluorescence progressively recovered to control levels, and co-localised with WGA, confirming targeting of the fusion protein to the apical membrane. Following incubation with TM, the levels of WT fluorescence at 3, 6 and 24 h were similar to that of untreated controls, but, with time, the predominant species of the protein shifted from the glycosylated (~70 kDa) form to the non-glycosylated (~67 kDa) form, confirmed by SDS-PAGE and Western blotting (Ortega et al. 2002). Nevertheless, the fusion protein was still expressed at the apical membrane. Transient expression of MDCK II cells with either WT or N98Q, resulted in fluorescence predominantly at the apical pole of the cells, colocalising with PNA. We conclude that polarized membrane targeting of Kir1.1b is unaffected by either prevention of the addition of complex oligosaccharides by tunicamycin, or by elimination of the N-linked glycosylation motif, when expressed in either form of MDCK cell type. These findings indicate that Kir1.1b protein is transported to the apical membrane via a mechanism(s) independent of glycosylation of the channel per se.