Two electrophoreticforms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrro gigas have been detected by Western analysis The faster movmg form co-migrates with the large subunit from punfied, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subumt from purified hydrogenase revealed that It is 15 amino acids shorter than that predIcted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between H~s"~ and Val"'. residues which are highly conserved among [NiFe] hydrogenases.Mutagenesis of the analogous residues, Hls5*' and Val"', m the E co/i hydrogenase-1 (HYDI) large subunit resulted m significant decrease in processing and HYDl activity.Hydrogenase large subunit; C-Terminal processmg; Mutagenesis