From the soybean cDNA library, we isolated and analyzed the chlH gene encoding a subunit of Mg-chelatase. The subunit was a polypeptide of 1,383 amino acids with a molecular mass of 153,491 Da, which shared 90% identity with the olive gene from Antirrhinum majus. The regulation of the expression of chlH was investigated in photomix-otrophic soybean suspension cells (SB-P). The expression was light-inducible, and the induction was more rapid than those of chlI and cab2. Furthermore, the levels of the transcripts and products of chlH appeared to be regulated by a circadian oscillation. The subchloroplastic localization of ChlH was investigated by immunoblot analyses with antiserum against recombinant ChlH. Depending on the concentration of Mg2+ in the lysis buffer, the localization of ChlH protein migrated between the stroma and the envelope membrane; ChlH was localized on the envelope membrane, a major site of chlorophyll biosynthesis, when the Mg2+ concentration of the lysis buffer was high (above 5 mM). These results indicated that the activity of Mg-chelatase was regulated by modulation of the expression and subchloroplastic localization of ChlH protein.