We have shown previously that during in vitro import into chloroplasts, the precursor of the major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and .apprx.26 kDa) which are inserted into the thylakoids. However, during incubation of the LHCP precursor with a chloroplast-soluble extract in an organelle-free processing reaction, the NH2 terminus is cleaved, yielding only a 25-kDa peptide. In the present study, mutations at the transit peptide-mature protein junction were introduced in the LHCP precursor to investigate the relationship between the two peptides and the determinants of proteolytic processing. Mutant p.DELTA.3 lacks 3 amino acids including Met34 at the primary cleavage site thought to give rise to the 26-kDa peptide. It is still processed during import and in the organelle-free reaction yielding in both assays only a 25-kDa peptide. Mutant p+4 has 4 amino acids inserted immediately after Met34 and a proline that disrupts the .alpha.-helix predicted by the Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120) to extend through this region. Although p+4 is imported, it is inefficiently processed; both a 25- and 26-kDa peptide are found, but at least 60% of the imported precursor remains uncleaved. Less than 5% is processed in the organelle-free assay. Replacement of the predicted .alpha.-helix in the mutant p+4.alpha. restores processing upon import into the chloroplast, yields only a 26-kDa peptide. p+4.alpha. is not processed in the organelle-free reaction. These results provide evidence that the two forms of LHCP obtained during import are the result of independent processing at two cleavage sites: the first site at Met34, and a second .apprx.10 amino acids downstream within what has been designated the NH2 terminus of the mature protein. Whereas p.DELTA.3 has the first site removed but retains a functional second site, in p+4.alpha. only the first site, or one very near it, is accessible to the processing enzyme during import. The conditions of the organelle-free reaction are specific for processing at only the secondary site. We discuss the implications of these findings in terms of the heterogeneity of LHCP in vivo.