The plasma membrane in plant cells is energized with an electrical potential and proton gradient generated through the action of H ⁺ pumps belonging to the P-type ATPase superfamily. The Arabidopsis genome encodes 11 plasma membrane H ⁺ pumps. Auto-inhibited H ⁺ -ATPase isoform 10 (AHA10) is expressed primarily in developing seeds. Here we show that four independent gene disruptions of AHA10 result in seed coats with a transparent testa ( tt ) phenotype (light-colored seeds). A quantitative analysis of extractable flavonoids in aha10 seeds revealed an ≈100-fold reduction of proanthocyanidin (PA), one of the two major end-product pigments in the flavonoid biosynthetic pathway. In wild-type seed coat endothelial cells, PA accumulates in a large central vacuole. In aha10 mutants, the formation of this vacuole is impaired, as indicated by the predominance of multiple small vacuoles observed by fluorescence microscopy using a vacuole-specific dye, 5-(and -6)-carboxy 2′,7′-dichlorofluorescein diacetate. A similar vacuolar defect was also observed for another tt mutant, tt12 , a proton-coupled multidrug and toxic compound extrusion transporter potentially involved in loading provacuoles with a flavonoid intermediate required for PA production. The endothelial cells in aha10 mutants are otherwise healthy, as indicated by the lack of a significant decrease in ( i ) the accumulation of other flavonoid pathway end products, such as anthocyanins, and ( ii ) mRNA levels for two endothelium-specific transcripts ( TT12 and BAN ). Thus, the specific effect of aha10 on vacuolar and PA biogenesis provides genetic evidence to support an unexpected endomembrane function for a member of the plasma membrane H ⁺ -ATPase family.