Citrus fruits are rich in carotenoids. In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYb, EC:1.14.-.-) is a key regulatory enzyme in the catalysis of lycopene to β-carotene, an important dietary precursor of vitamin A for human nutrition. Two closely related lycopene β-cyclase cDNAs, designated CsLCYb1 and CsLCYb2, were isolated from the pulp of orange fruits (Citrus sinensis). The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopeneaccumulating genotype Cara Cara than that in common genotype Washington, and this might be correlated with lycopene accumulation in Cara Cara fruit. The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system, but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene. In tomato transformation studies, expression of CsLCYb1 under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene, and the ripe fruits displayed a bright orange colour. However, the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation. In fruits of the CsLCYb1 transgenic plants, most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g-1 DW. Unexpectedly, most transgenic tomatoes showed a reduction in total carotenoid accumulation, and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits. Collectively, these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency, and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.