The ability to differentiate between ions is a property of ion channels that is crucial for their biological functions’, However, the fundamental structural features that define anion selectivity and distinguish anion-permeable from cation-permeable channels are poorly understood, Voltage-gated chloride (Cl-) channels belonging to the ClC family are ubiquitous and have been predicted to play important roles in many diverse physiological(2) and pathophysiological(3-5) processes. We have identified regions of a human skeletal muscle ClC isoform that contribute to formation of its anion-selective conduction pathway. A core structural element (pi region) of the CIC channel pore spans an accessibility barrier between the internal and external milieu, and contains an evolutionarily conserved sequence motif : GKxGPxxH. Neighbouring sequences in the third and fifth transmembrane segments also contribute to isoform-specific differences in anion selectivity, The conserved motif in the Cl- channel Pl region may constitute a ’signature’ sequence for an anion-selective ion pore by analogy with the homologous GYG sequence that is essential for selectivity in voltage-gated potassium ion (K+) channel pores(6-8).