The biochemical basis of color as an aesthetic quality in mature fruit of navel and Valencia orange (Citrus sinensis) was determined. Saponification of the two major color-imparting components resolved by thin-layer chromatography, followed by reversed-phase high-performance liquid chromatography, revealed that these comprised acyl esters of (9Z)-violaxanthin and beta-citraurin. Identification of the chromophores was based on cochromatography and online spectral analysis. The color quality of flavedo of mature fruit was dependent on the content and relative amounts of (9Z)-violaxanthin and beta-citraurin. Quantitative results revealed that increased color intensity was associated with a decline in the (9Z)-violaxanthin:beta-citraurin ratio from greater than 50 to below 10, an increase in flavedo (9Z)-violaxanthin and beta-citraurin content, and that measurement of the mass and ratio of these carotenoids can be used to accurately color-grade orange fruit for local and export markets.