Carotenoids are the main pigments responsible of the colouration of Citrus fruits. The beta-cyclization of lycopene, catalysed by the lycopene beta-cyclases (beta-LCY), seems to be a key regulatory step of the carotenoid pathway. In the present study, two beta-LCYs from orange fruits (Citrus sinensis), named Csbeta-LCY1 and Csbeta-LCY2 have been isolated and the activity of the encoded proteins was demonstrated by functional analysis. Csbeta-LCY1 was expressed at low levels and remained relatively constant during fruit ripening while Csbeta-LCY2 showed a chromoplast-specific expression and a marked induction in both peel and pulp of orange fruits in parallel with the accumulation of beta,beta-xanthophylls. The potential involvement of Csbeta-LCY2 in the accumulation of lycopene, characteristic of some Citrus species such as red grapefruits, was investigated. Expression of Csbeta-LCY2 and another seven carotenoid biosynthetic genes were studied in the peel and pulp of the high lycopene-accumulating grapefruit, Star Ruby, and compared with those of ordinary Navel orange. In Star Ruby, the accumulation of lycopene during fruit maturation was associated with a substantial reduction in the expression of both beta-LCY2 and beta-CHX genes with respect to Navel orange. Moreover, two different alleles of beta-LCY2: beta-LCY2a and beta-LCY2b were isolated from both genotypes, and functional assays demonstrated that the lycopene beta-cyclase activity of the allele b was almost null. Interestingly, Star Ruby grapefruit predominantly expressed the unfunctional beta-LCY2b allele during fruit ripening whereas Navel oranges preferably expressed the functional allele. It is suggested that the presence of diverse alleles of the beta-LCY2 gene, encoding enzymes with altered activity, with different transcript accumulation may be an additional regulatory mechanism of carotenoid synthesis involved in the accumulation of lycopene in red grapefruits.