In vitro import of the precursor of tobacco cytochrome f, which is is encoded by the chloroplast genome, into isolated pea thylakoids was analyzed. Upon incubation with thylakoids and a stromal fraction, the precursor of cytochrome f was efficiently imported into thylakoids. The imported cytochrome f was tightly integrated into the thylakoid membrane. Insertion of cytochrome f into the thylakoid membrane was blocked by nigericin, sodium azide, and antibodies against pea chloroplast SecA. These results suggest that cytochrome f utilizes the bacterial-type SecA-dependent pathway to be imported into thylakoids. (C) 1996 Academic Press, Inc.