A bacterial homolog of the mammalian mitochondrial benzodiazepine receptor, the tryptophan-rich sensory protein (TspO) has been previously demonstrated to negatively affect the transcriptional expression of several photosynthesis genes of Rhodobacter sphaeroides. To identify components of the signal transduction pathway from the outer membrane-localized TspO to the DNA-active transcription factor(s), we examined the involvement of TspO in the regulation of tetrapyrrole metabolism in R. sphaeroides. By analyzing the tetrapyrrole pigments accumulated by resting cell suspensions of R. sphaeroides, we demonstrated that TspO negatively regulates the activity of coproporphyrinogen III oxidase in this bacterium. hemN, encoding one of the isoenzymes of coproporphyrinogen III oxidase of R. sphaeroides, provided in trans to the wild type strain, produced a TSPO1 mutant phenotype by abolishing the negative effect of TspO on the transcription of the photosynthesis genes, crtI and puc. It is proposed that TspO, by regulating the exit of certain tetrapyrrole intermediates of the heme/bacteriochlorophyll biosynthetic pathways in R. sphaeroides in response to the availability of molecular oxygen, causes the accumulation of a biosynthetic intermediate that serves as a corepressor for both specific pigment gene transcription and the puc operon. The relationship between the bacterial TspO and the mitochondrial peripheral benzodiazepine receptor is discussed.