We report on the isolation, functional expression and characterization of a cDNA encoding chlorophyllase, the enzyme catalyzing the first step in the chlorophyll breakdown pathway. The Chlase1 cDNA from Valencia Orange (Citrus sinensis cv. Valencia) was obtained by RT-PCR using degenerate primers based on the amino acid sequence of the previously purified protein. Chlase1 encodes a protein of 329 amino acids, including a sequence domain characterizing serine-lipases and a putative chloroplast-directing transit peptide. The Chlase1 gene encodes an active chlorophyllase enzyme which catalyzes the dephytylation of chlorophyll as shown by in vitro recombinant enzyme assays. Chlorophyllase expression at the transcript level in Valencia orange peel was found to be low and constitutive during natural fruit development without significant increase towards color-break and ripening. However, ethylene treatment induced an increase in chlorophyllase transcript at all stages of development. An enhanced response to ethylene treatment was observed during the months of October and November, corresponding to the time of natural color-break. The senescence-delaying regulator gibberellin-A3 (GA3) inhibited the effect of ethylene on chlorophyllase transcript accumulation. The data presented suggest that chlorophyllase may not be the regulator of chlorophyll breakdown during natural fruit ripening but is consistent with the notion that chlorophyll is gradually degraded during ripening due to a negative balance between synthesis and breakdown. According to this model, exogenous application of ethylene accelerates chlorophyll breakdown due to increased de novo synthesis of chlorophyllase. Further experimentation on the regulation and role of chlorophyllase in planta will be facilitated by the gene tools established in this work.