Cell-free systems from Corynebacterium poinsettiae and Micrococcus luteus incorporated labeled mevalonic acids into acyclic C40 and cyclic C50 carotenoids. When (3R,4R)-[2-14C,4-3H1]mevalonate was used as substrate, the 14C:3H ratios of C.p.450 and sarcinaxanthin showed that the hydrogen atoms at C-2 of both carotenoids, and that at C-6 of sarcinaxanthin, are derived from the 4-pro-R position of mevalonate. The 14C:3H ratios of C.p.450 and sarcinaxanthin synthesized from (2RS,3R)-[2-14C,2-3H2]mevalonate showed that both hydrogen atoms of C-4 are derived from those at C-2 of mevalonate. These results exclude epsilon- and beta-rings as precursors of the gamma-ring. They also exclude the interconversion of the epsilon- and beta-rings. Sarcinaxanthin samples synthesized from (3R,4R)-[2-14C,4-3H1]- and (2RS,3R)-[2-14C,2-3H2]mevalonate by a cell-free system from M. luteus were found to undergo isomerization in strong alkali. The major product of isomerization (85%) was decaprenoxanthin (epsilon-ring) with the beta-ring C.p.450 present in small amounts (3% yield). The 14C:3H ratios of these isomerization products were consistent with the loss of one C-4 hydrogen atom from each epsilon-ring of the former and one C-6 hydrogen atom from each beta-ring of the latter.