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 lycopeneβ-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 lfavedo and juice sacs of a lycopene-accumulating genotype Cara Cara than that in common genotype Washington, and this might be correlated with lycopene accumulation in Cara Cara fruit. The CsLCYb1 efifciently converted lycopene into the bicyclicβ-carotene in an Escherichia coli expression system, but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into theβ-carotene and the monocyclic γ-carotene. In tomato transformation studies, expression of CsLCYb1 under the control of the caulilfower mosaic virus (CaMV) 35S constitutive promoter resulted in a virtually complete conversion of lycopene intoβ-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 intoβ-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 efifciency, and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.