The open reading frames of tomato beta-galactosidase (TBG) 4 and 5 cDNAs were expressed in yeast, and the enzymes properties and substrate specificities were investigated. The two enzymes had peak activities between pH 4-4.5 and 37-45 degrees C. TBG4 specifically hydrolyzed beta-(1-->4) and 4-linked galactooligosaccharides. TBG5 had a strong preference to hydrolyze beta-(1-->3) and beta-(1-->6)-linked galactooligosaccharides. Exo-beta-galactanase activity of the TBG enzymes was measured by determining the release of galactosyl residues from native tomato cell wall fractions throughout fruit development and ripening. Both TBGs released galactose from all of the fractions and stages tested. TBG4 activity was highest using chelator soluble pectin and alkali soluble pectin at the turning stage of ripening. Using aminopyrene trisulfonate labeled substrates, TBG4 was the only enzyme with strong exo-beta-(1-->4)-galactanase activity on 5 mer or greater galactans. TBG4 and TBG5 were both able to degrade galactosylated rhamnogalacturonan. Neither enzyme was able to degrade galactosylated xyloglucan.