A preparation of hydrogenase from Wolinella (formerly Vibrio) succinogenes enriched in 33S to at least 70% has been studied by EPR spectroscopy. The sulphur isotope, which has a nuclear spin of 3 2 , clearly broadened the spectrum of the Fe-S cluster. Resolved hyperfine splitting was observed in one of the lines of the EPR spectrum of Ni(III). It was concluded that this was due to interaction with one 33S nucleus. Also the appearance of the high-field line of the EPR signal of Ni(I), either before or after photodissociation of the nickel-hydrogen bond, indicated hyperfine interaction with one 33S nucleus. No indication has been observed for nitrogen hyperfine interaction in any of the EPR spectra in this study. The data provide independent proof of earlier conclusions from EXAFS studies by two other groups (Lindahl, P.A., Kojima, N. Hausinger, R.P., Fox J.A., Teo, B.K., Walsh C.T. and Orme-Johnson, W.H. (1984) J. Am. Chem. Soc., 106, 3062–3064, and Scott R.A., Wallin, S.A., Czechowsky, M., DerVartanian, D.V., LeGall, J., Peck, H.D.,Jr. and Moura, I. (1984) J. Am. Chem. Soc. 106, 6864–6865) that sulphur is present in the direct coordination of nickel in hydrogenase. The EPR data provide evidence for the presence of only one, or possibly two, S ligands.