β-galactosidases have been detected in a wide range of plants and are characterized by their ability to hydrolyse terminal non-reducing β-D-galactosyl residues from β-D-galactosides. These enzymes have been detected in a wide range of plant organs and tissues. In a search for differentially expressed genes during the abscission process in citrus, sequences encoding β-galactosidase were identified. Three cDNA fragments of a β-galactosidase gene were isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after the application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-pyrazole). Based on sequence information derived from these fragments, a full-length cDNA of 2847 nucleotides (GenBank accession number AY029198) encoding β-galactosidase was isolated from mature fruit abscission zones by 5′- and 3′-RACE approaches. The β-galactosidase cDNA encoded a protein of 737 amino acid residues with a calculated molecular weight of 82 kDa. The deduced protein was highly homologous to plant β-galactosidases expressed in fruit ripening. Southern blot analysis demonstrated that at least two closely related β-galactosidase genes were present in ‘Valencia’ orange. Temporal expression patterns in mature fruit abscission zones indicated β-galactosidase mRNA was detected 48 h after treatment of CMN-pyrazole and ethephon in mature fruit abscission zones. β-galactosidase transcripts were detected in leaf abscission zones only after ethephon application. The citrus β-galactosidase was expressed in stamens and petals of fully opened flowers and young fruitlets. The results suggest that this β-galactosidase may play a role during abscission as well as early growth and development processes in flowers and fruitlets.