The homologous import and membrane association of a key enzyme for chlorophyll biosynthesis, the NADPH:protochlorophyllide (Pchlide) oxidoreductase (PAR, EC 1.6.99.1) into pea chloroplasts was investigated in vitro. The co-factor, NADPH, decreased binding of the precursor protein (pPOR) to the envelope membranes in the presence of ATP. The decrease of the binding reaction with NADPH was not observed with the precursor of the small subunit of Rubisco (pSS). To investigate possible substrate-dependency for the import reaction, internal Pchlide concentrations in the plastids were raised by either an addition of ÎŽ-aminolevulinic acid to isolated plastids or etiolation of the seedlings prior to plastid isolation. Increased amounts of plastid-bound Pchlide gave no observable differences in POR import. The capacity of POR and 11 different POR mutants, carrying charged-to-alanine scanning substitutions, to form a catalytically active POR-Pchlide-NADPH complex and to associate with the thylakoid membranes in a protease-resistant way were tested. Wild-type POR, as well as the mutants with charge substitutions in the N-terminal region of the protein, exhibited higher catalytic activity than the POR mutants carrying substitutions in the C-terminal region. Formation of a catalytically active complex did not, however, increase the association efficiency onto the thylakoids. We can, therefore, postulate that the import of pea POR into pea chloroplasts was not substrate-dependent, nor did formation of catalytically active complexes stimulate or inhibit the membrane association reaction of POR.