Changes in the biosynthesis of individual carotenoid pigments have been investigated during fruit ripening of five cultivars of red pepper (Capsicum annuum L.): Mana, Numex, Belrubi, Delfin, and Negral (a chlorophyll-retaining mutant when ripe). The study was carried out throughout the ripening process, and with special emphasis on the ripe stage, to discover possible differences between cultivars and to characterize these by their carotenoid pattern and content for selecting the best varieties for breeding programs. Ripening fruit of the five cultivars showed the typical and characteristic pattern of carotenoid biosynthesis for the Capsicum genus. In the five cultivars, lutein and neoxanthin, both characteristic chloroplast pigments, decreased in concentration with ripening and eventually disappeared. beta-Carotene, antheraxanthin, and violaxanthin increased in concentration, and other pigments were biosynthesized de novo: zeaxanthin, beta-cryptoxanthin, capsanthin, capsorubin, capsanthin-5,6-epoxide, and cucurbitaxanthin A. A pool of zeaxanthin stands out of the rest of pigment during ripening, which reveals the importance of this pigment as a branching point in the carotenoid biosynthesis in Capsicum. Quantitatively, Negral cultivar showed the highest increase in total carotenoid content (48. 39-fold), followed by Mana and Delfin with 38.03- and 36.8-fold, respectively, and by Belrubi and Numex with 28.03- and 23.48-fold, respectively. In all the red varieties, there was an inverse relationship between total carotenoid content and the red to yellow isochromic pigment fraction ratio (R/Y) and the capsanthin-to-zeaxanthin ratio (Caps/Zeax). This seems to be related to the carotenogenic capacity of the cultivar, and thus selection and breeding should not only seek a higher total carotenoid content but also attempt to increase these ratios. In the present study, the cultivar Mana had the highest total carotenoid content (13 208 mg/kg dwt), but the lowest R/Y (1.25) and Caps/Zeax (3.38) ratios, which are therefore the parameters to improve. The cultivar Negral had a high carotenoid content (8797 mg/kg dwt) and high R/Y and Caps/Zeax ratios and could be used for transfer of these characters in direct crosses with the cultivar Mana. The cultivar Numex had the highest Caps/Zeax ratio (7.17) and is thus an ideal progenitor for this character.