Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of orthophosphate (Pi)-starved (-Pi) plants. APases catalyze Pi hydrolysis from a broad range of phosphomonoesters at an acidic pH. The largest class of nonspecific plant APases is comprised of the purple APases (PAPs). Although the biochemical properties, subcellular location, and expression of several plant PAPs have been described, their physiological functions have not been fully resolved. Recent biochemical studies indicated that AtPAP26, one of 29 PAPs encoded by the Arabidopsis (Arabidopsis thaliana) genome, is the predominant intracellular APase, as well as a major secreted APase isozyme up-regulated by -Pi Arabidopsis. An atpap26 T-DNA insertion mutant lacking AtPAP26 transcripts and 55-kD immunoreactive AtPAP26 polypeptides exhibited: (1) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (2) a 40% decrease in secreted APase activity during Pi deprivation, (3) 35% and 50% reductions in free and total Pi concentration, respectively, as well as 5-fold higher anthocyanin levels in shoots of soil-grown -Pi plants, and (4) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function occurred under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. Transient expression of AtPAP26-mCherry in Arabidopsis suspension cells verified that AtPAP26 is targeted to the cell vacuole. Our results confirm that AtPAP26 is a principal contributor to Pi stress-inducible APase activity, and that it plays an important role in the Pi metabolism of -Pi Arabidopsis.