We have characterized the carotenoids accumulated by a series of mutants of Rhodopseudomonas capsulata as part of a study of the synthesis, structure, and function of the photosynthetic membranes of this bacterium. The carotenoids in this study were identified by visible and mass spectroscopy, chromatography, derivatization, and chemical analyses. We have located a new genetic region, crtF, necessary for the O-methylation of the carotenoids. Mutants with a lesion in crtF accumulate demethylspheroidene as their major carotenoid during anaerobic growth and demethylspheroidenone when grown in the presence of oxygen, a heretofore undescribed phenotype. The genetic region necessary for O-methylation maps adjacent to the known cluster of genes affecting carotenoid biosynthesis. In addition, we have identified methoxyneurosporene as the carotenoid that preferentially binds to the reaction centers of strain Ga, a green mutant of R. sphaeroides which accumulates three neurosporene-like carotenoids. A metabolic grid for carotenoid biosynthesis is proposed, based upon the intermediates accumulated in these mutants.