Quantitative carotenoid analyses of 3 additional species representing each of three pigment types of Prasinophyte algae grown in pure culture are reported. The carotenoids were characterized by chromatographic (TLC, HPLC), spectroscopic (visible and mass spectroscopy, and, in part, ¹H NMR, circular dichroism), and chemical methods. Preprasinoxanthin was isolated for the first time and the structure of 6"-hydroxy siphonaxanthin 19-(trans-Δ²-dodecenoate) was elucidated. The presence of siphonein, identified as siphonaxanthin 19-(trans-Δ²-dodecenoate) in Prasinophyceae was confirmed. Small amounts of the corresponding trans-Δ²-decenoates were present. Authentic siphonaxanthin 19-(trans-Δ²-dodecenoate) was isolated from Codium fragile for comparison. Pyramimonas amylifera belonged to the siphonein type. Prasinococcus capsulatus produced carotenoids of the prasinoxanthin/uriolide type, whereas Nephroselmis olivacea only contained common green algal carotenoids. A chemosystematic evaluation is made on the basis of 13 species examined by methods including mass spectroscopy and supplemented by less documented literature data on 26 species. Plausible biosynthetic routes are proposed from structural interrelationships, including chiralities. The Prasinophyceae displays a diversified carotenoid complement; 30 carotenoids have been identified, 14 of which are peculiar to this algal class. Three carotenoid prototypes emerge: type 1, producing only common green algal type carotenoids; type 2 with additional carotenoids of the siphonaxanthin series; and type 3 with additional carotenoids of the prasinoxanthin/uriolide series. Carotenoids with ε- and γ-end groups are abundant. The 7,8-dihydro feature of the unique uriolide series is compatible with unprecedented cyclization of the least unsaturated end group of neurosporene by an ε-cyclase.