Micrococcus roseus forms bicyclic keto-carotenoids. The effects of nicotine, piperonyl butoxide, and 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) were studied with regard to their ability to selectively inhibit carotenogenesis in the organism. Nicotine caused accumulation of beta-zeacarotene; piperonyl butoxide caused accumulation of phytoene and traces of phytofluene, zeta-carotene, and beta-zeacarotene. In both cases canthaxanthin biosynthesis was inhibited. CPTA inhibited canthaxanthin synthesis and caused accumulation of beta-zeacarotene and gamma-carotene and their mono- and di-hydroxy derivatives. Regardless of the inhibitor used, canthaxanthin was the major colored carotenoid biosynthesized. The expected precursors of carotenoid cyclization, neurosporene and (or) lycopene, were not detected in CPTA- or nicotine-inhibited cultures. Therefore, carotenoid cyclization in M. roseus does not involve neurosporene or lycopene and must occur early in carotene biosynthesis, prior to the formation of beta-zeacarotene, zeta-Carotene is proposed as the cyclization substrate and beta-zeacarotene as the substrate for oxygen insertion.