A combination of limited tryptic proteolysis, reverse phasehigh performance liquid chromatography, Edman degradative sequencing, amino acid analysis, and fast-atom bombardment mass-spectrometry was used to remove and identify the first 14 to 18 N-terminal amino acid residues of the large subunit of higher plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas relnhardtii, Marchantia polymorpha, pea (Pisum satvum), tomato (Lycoperskon esculentum), potato (Solanum tuberosum), pepper (Capskum annuum), soybean (Glycine max), petunia (Petunia x hybrida), cowpea (Vigna sinensis), and cucumber (Cucumis sativus) plants. The N-terminal tryptic peptide from acetylated Pro-3 to Lys-8 of the large subunit of Rubisco was identical in all species, but the amino acid sequence of the penultimate N-terminal tryptic peptide varied. Eight of the 10 species examined contained a trimethyllysyl residue at position 14 in the large subunit of Rubisco, whereas Chlamydomonas and Marchantla contained an unmodified lysyl residue at this position.Rubisco (EC 4.1.1.39) is a large hexadecameric protein, with 8 large and 8 small subunits, that catalyzes the fixation of atmospheric CO2 during photosynthesis (2). The large subunit is encoded by chloroplast DNA (8) and the small subunit by nuclear DNA (13). The small subunit is synthesized as a precursor with an N-terminal transit sequence that targets the polypeptide for import into the chloroplast. The transit sequence is proteolytically removed pnor to assembly ofsmall subunits with large subunits by chloroplast chaperonins (9). Synthesis of the large subunit in the chloroplast is also followed by posttranslational processing (16,25). The Nterminal Met-and Ser-2 are removed and Pro-3 acetylated in Rubisco from spinach (Spinacia oleracea L.), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), and muskmelon (Cucumis melo). Additionally, the tobacco and musk-'