␣-l-Arabinofuranosidases (␣-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different ␣-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesissuppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. ␣-Af I and II are active throughout fruit ontogeny. ␣-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. ␣-Af II activity accounts for over 80% of the total ␣-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, ␣-Af III is ethylene dependent and specifically active during ripening. ␣-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas ␣-Af II and III acted on Na 2 CO 3 -soluble pectins. Different ␣-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. ␣-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only ␣-Af III activity. Results suggest that tomato ␣-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.Fruit differentiation, growth, and ripening depend on changes in the architecture of cell walls. These processes involve the modification of the amount and composition of pectic and hemicellulosic polysaccharides, which takes place as a coordinated series of assembly and disassembly steps. The removal of side chains from the backbones of different matrix polysaccharides is attributable to the action of glycosidases (Fry, 1995), but the actual role of these enzymes in vivo and their regulation remain unknown. In the last few years, considerable attention has been given to the release of neutral sugars from the cell wall, a major process in the development and ripening of tomato (Lycopersicon esculentum) fruit, and to understanding how the various enzymes and their different isoforms affect these processes. A multigenic ␤-galactosidase (␤-Gal) gene family has recently been identified (Smith and Gross, 2000); three different gene products were purified and characterized for the first time two decades ago (Pressey, 1983). One of them, designated ␤-Gal II, is specifically active during ripening and is probably involved in fruit softening (Pressey, 1983;Carey et al., 1995;Smith et al., 1998;Sozzi et al., 1998). However,...