The biosynthetic pathway of monocyclic carotenoids in the yeast Phaffia rhodozyma was studied by identifying carotenoids, applying inhibitors of carotenoid synthesis, and analyzing the carotenoids in carotenogenic mutants. Two carotenoids, torulene and 3,3'-dihydroxy-beta,phi-carotene-4,4'-dione (DCD), were identified from the yeast. Piperonyl butoxide inhibited dehydrogenation of carotenes and caused accumulation of neurosporene, lycopene, gamma-carotene, and beta-zeacarotene. Yellow mutants of P. rhodozyma produced mainly beta-carotene, which is a dicyclic carotene produced from neurosporene through lycopene. The yellow mutants grown with 0.01% triethylamine (TEA) or 2 mM 2-methylimidazole (MI) produced significantly increased amounts of torulene, which is a monocyclic carotene produced from neurosporene through beta-zeacarotene. When red strains of P. rhodozyma were grown with TEA or MI, they produced increased amounts of beta-zeacarotene, torulene, and 3-hydroxy-3',4'-didehydro-beta,phi-carotene-4-one (HDCO) (all monocyclic). DCD, accumulated especially in old cultures, was also increased in MI or TEA cultures coincident with the increases in HDCO. We propose the following monocyclic carotenoid pathway in P. rhodozyma: neurosporene-->beta-zeacarotene-->gamma-carotene-->torulene-->HDCO-->DCD.