A 2.4 kilobase-pair segment of the spinach plastid chromosome carrying the genes for apocytochrome b6 and subunit 4 of the thylakoid membrane cytochrome b/f complex has been analysed by DNA sequencing and Northern blot analysis. The nucleotide sequence reveals two uninterrupted open reading frames of 211 and 139 triplets coding for two hydrophobic proteins of 23.7 kd (cytochrome b6) and 15.2 kd (subunit 4). The genes are located on the same strand and are separated from each other by 1018 untranslated base pairs. They map adjacent to the gene for the P680 chlorophyll α apoprotein of the photosystem II reaction center. The three genes appear to be under common transcriptional control and the transcripts post-transcriptionally modified. The deduced amino acid sequences of cytochrome b6 and subunit 4 both exhibit significant homology with published sequences from mitochondrial b cytochromes (42 kd) suggesting that these functionally equivalent polypeptides in photosynthetic and respiratory electron transport chains arose monophyletically.