The cellular location of membrane-bound NiFe-hydrogenase 2 (HYD2) from Escherichia coli was studied by immunoblot analysis and by activity staining. Treatment of spheroplasts with trypsin was able to release active HYD2 into the soluble fraction, indicating that HYD2 is attached to the periplasmic side of the cytoplasmic membrane and that HYD2 undergoes a trans-membrane translocation during its biosynthesis. By using a nik mutant deficient in the high affinity specific nickel transport system, we show that the intracellular availability of nickel is essential for the processing of the large subunit and for the transmembrane translocation of HYD2. We also demonstrate that the processing of the precursor, which is related with nickel incorporation, can occur in the membrane-depleted soluble fraction and that it is associated with the increase in resistance to proteolysis of the processed form of the large subunit. The mechanism of the transmembrane translocation of HYD2 is discussed.