The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) gene, which encodes the Ca2+ channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits an unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer 45Ca2+ influx assay and the voltage-clamp technique using Xenopus laevis oocytes. Among the variations tested, including A8V, R154H, A563T, and L712F, the latter two significantly increased TRPV5-mediated Ca 2+ influx. The A563T variant, which exists in African Americans with relative high frequency, exhibited increased Ca2+ influx at extracellular Ca2+ from 0.01 to 2 mM despite a lower expression level at the plasma membrane. This variant also exhibited a reduction in Na + current as a result of increased sensitivity to extracellular Mg2+. By substituting threonine-563 (Thr563) with serine or valine residue, the bulky side chain of Thr563 was shown to facilitate Ca2+ transport, whereas the hydroxyl group of Thr 563 is likely related to Mg2+ sensitivity. The A563T variant was capable of increasing TRPV5-mediated Ca2+ influx, even when it was expressed under conditions mimicking heterozygous or compound state with other variants. In conclusion, the A563T variant of TRPV5 significantly increased Ca2+ influx by affecting the Ca2+ permeation pathway. Thus the A563T variation in TRPV5 may contribute to the superior ability of renal Ca2+ conservation in African Americans. Copyright © 2009 the American Physiological Society.