Mutations of the human SLC26A4/PDS gene constitute the most common cause of syndromic and non-syndromic hearing loss. Definition of the SLC26A4 mutation spectrum among different populations with sensorineural hearing loss is important for development of optimal genetic screening services for congenital hearing impairment. We screened for SLC26A4 mutations among Chinese and U.S. subjects with hearing loss using DHPLC and direct DNA sequencing. 52 of 55 Chinese subjects with deafness accompanied by enlargement of the vestibular aqueduct (EVA) exhibited at least one mutant SLC26A4 allele, whereas among 116 deaf Chinese patients without EVA SLC26A4 mutations were found in only two subjects. The spectrum of SLC26A4 mutations differed among Chinese and U.S. subjects, and included 10 previously unreported SLC26A4 variants: 4 in the Chinese population (p.E303Q, p.X329, p.X467, p.X573), and 6 in the U.S. population (p.V250A, p.D266N, p.F354S, p.D697A, p.K715N, p.E737D). Among the seven novel in-frame missense mutations, five encoded SLC26A4 proteins with substantially reduced Cl^-/anion exchange activity as expressed and measured in Xenopus oocytes, but four of these were sufficiently active to allow study of anion selectivity. The only mutant polypeptide exhibiting complete loss of anion exchange function, p.E303Q, was expressed at or near the oocyte surface at near-wildtype levels. Two variants, p.F354S and p.E737D, displayed selective reduction in the relative rate of Cl^-/HCO₃^- exchange compared to similarly measured rates of Cl^-/Cl^- exchange and Cl^-/I^- exchange. Our data show that mutation analysis of the SLC26A4 gene is of high diagnostic yield among subjects with deafness and bilateral EVA in both China and the U.S. However, the pathogenicity of monoallelic SLC26A4 gene variants in patients with hearing loss remains unclear in many instances.