A 1.6 kbp DNA segment of spinach plastid DNA has been shown to carry the gene for the proteolipid subunit of the ATP synthase. Each plastid chromosome contains one copy of this gene which is located in the large single-copy region of the chromosome near that of the ATP synthase alpha subunit. These two genes are transcribed in the same direction and probably in distinct RNA species. The proteolipid gene was located by hybrid-selection mapping, by transcription/translation of recombinant DNAs and by nucleotide sequencing. The in vitro product was identified by electrophoretic criteria including its characteristic shift in electrophoretic mobility upon incubation with dicyclohexylcarbodiimide, and immunology. The nucleotide sequence of the proteolipid gene is uninterrupted. The deduced amino acid sequence coincides with the published amino acid sequence for this protein and shows little homology with the published sequence of the proteolipid subunit of E. coli.