The active centers of NiFe hydrogenase from Desulfovibrio vulgaris Miyazaki F has been located in the electron density map calculated at 4 Å resolution. The electron density map based on five heavy atom derivatives showed four strong peaks which were clearly distinguished from the protein region. These strong densities have been successfully assigned to three iron‐sulfur clusters and one nickel atom by difference Fourier technique with coefficients of the best phases from multiple isomorphous replacement (MIR) method and structure factors obtained at five wavelengths (1.040, 1.487, 1.730, 1.743, and 1.750Å) with use of the synchrotron radiation source． Four active centers are approximately lined up with a distance of ca．13Å，which seems to be reasonable for them to be connected with electron transfer chain.