The isolation of a cytochrome f/b6 complex from spinach chloroplasts, with high yield and purity is reported. The complex consists of five polypeptides with a molecular mass of 34, 33, 23.5, 20 and 17.5 kDa, and contains one cytochrome f, two cytochromes b6 and the Rieske Fe-S center with two non-heme irons. It does not contain plastocyanin and is almost completely devoid of chlorophyll and carotenoids, but lipid and detergent are present. It is lacking cytochrome b-559, although three of the five polypeptides seem to carry heme groups. The preparation has plastoquinol-plastocyanin oxidoreductase activity with plastoquinol-1 and plastoquinol-9, which is sensitive to 2,5-dibromomethylisopropyl-p-benzoquinone, to 2-iodo-6-isopropyl-3-methyl-2',4',4'-trinitrodiphenyl ether, to 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, and to bathophenanthroline. Characteristics of this activity with respect to substrate concentrations, pH, detergent effect and other parameters are described.