Mutations in the CLCNKB gene encoding the CLC-Kb, kidney chloride channel, are the cause of classic Bartter syndrome (BS) type III. This is an autosomal recessive disease characterized by salt-wasting tubulopathy and associated with secondary hyperaldosteronism, hypokalaemic metabolic alkalosis and normal blood pressure (1). To understand better the implication of ClC-Kb channels in the development of BS is necessary to deepen the knowledge on mutated forms of ClC-Kb. In this study we investigate the functional consequences of 2 novel (L81P and G246R) and 3 previously reported CLC-Kb homozygous missense mutations (R92W, A204T and R438H) (2, 3, and 4). By using X. laevis oocytes as heterologous expression system we functionally characterize the ClC-Kb mutants. Surface expression and Cl- currents data were collected for each mutant. Results are shown as mean ± SEM. Experiments included at least 8 measurements with 3 different batches of oocytes. Significance was analyzed with one-way ANOVA follows by Holm-Sidak test. P < 0.05 was considered significant. The mutants fell into 2 categories, those carrying no current: G246R (0.8±0.1 µA), R438H (0.6±0.1 µA) and those showing a residual conductance, with reduction by 30%, R92W (2.9 ±0.2 µA) or 60 %: L81P (1.6±0.2 µA) and A204T (1.3±0.3 µA) as compared to CLC-Kb WT (5.4±0.5 µA). Overall, the reduction in surface expression, estimated by chemiluminescence assay (oocytes), corresponded to a decrease in current amplitude for the different mutants. Functionally we investigated the sensitivities to external H+ and Ca2+, two characteristic properties of CLC-Kb, in mutants showing residual activity. We observed no difference in half-maximal pH inhibition (pKa) for L81P (7.74±0.67), R92W (7.77±0.13) and R351P (7.78±0.40) compared to WT (7.82±0.51). In contrast, there was a dramatic alteration in inhibition by H+ for A204T (6.72±0.01). No change in the slope factor was shown for any mutants respect to the WT. The stimulating effect of external Ca2+ was largely blunted. Altogether, our results show that reduced targeting to the membrane and/or lower stability of the membrane protein is the main functional consequence of these mutations. Additionally, the altered regulation of A204T would tend to alleviate the phenotypic consequences of the impairment of membrane expression by maintaining higher activity of the channel under physiological conditions. From a mechanistic point of view, the dramatic functional effects of this last mutation underscores a major role in CLC-Kb gating for this region which have never been described before.