Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen of the chloroplast. Maturation of preplastocyanin involves successive cleavages by a stromal peptidase and a distinct thylakoidal peptidase. In this report we have analyzed the precise location and orientation of the thylakoidal peptidase with respect to the thylakoid membrane. Experiments involving differential centrifugation of thylakoid extracts and sonication of isolated vesicles indicate that the peptidase is tightly bound to the thylakoid membrane but not intimately associated with any of the major thylakoid protein complexes. Analysis of the lateral distribution of the peptidase has shown that the enzyme is exclusively located in the non-appressed lamellae of the thylakoid network. The active site of the peptidase is on the lumenal face of the thylakoid membrane.