An acid phosphatase (APase, EC 3.1.3.2) from ripened banana (Musa cavendishii L. cv. Cavendish) fruit has been purified 1,876-fold to electrophoretic homogeneity and a final p-nitrophenylphosphate (pNPP)-hydrolyzing specific activity of 745 micromol Pi produced (mg protein)(-1) min(-1). Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with APase activity. SDS-PAGE and analytical gel filtration demonstrated that the purified enzyme exists as a 40-kDa monomer. That the enzyme is glycosylated was indicated by its tight absorption to Concanavalin A-Sepharose. Banana APase was relatively heat stable, displayed a symmetrical pH/activity profile with maximal activity at pH 5.8, and was activated 180% and 150% by 5 mM Mn2+ and Mg2+, respectively. The enzyme exhibited a broad substrate selectivity, with maximal specificity constants (Vmax/Km) obtained with pNPP, phosphoenolpyruvate, phenyl phosphate, and O-phospho-L-tyrosine. Potent inhibition by Pi, molybdate, vanadate, arsenate, and Zn2+ was observed. Putative metabolic functions of the APase are discussed in relation to maintaining significant Pi mobility during banana fruit ripening.