The iron-sulfur-cluster-free hydrogenase Hmd (H-2-forming methylenetetrahydromethanopterin dehydrogenase) from methanogenic archaea has recently been found to contain one iron associated tightly with an extractable cofactor of yet unknown structure. We report here that Hmd contains intrinsic CO bound to the Fe. Chemical analysis of Hmd revealed the presence of 2.4 +/- 0.2 mol of CO/mol of iron. Fourier transform infrared spectra of the native enzyme showed two bands of almost equal intensity at 2011 and 1944 cm(-1), interpreted as the stretching frequencies of two CO molecules bound to the same iron in an angle of 90degrees. We also report on the effect of extrinsic (CO)-C-12, (CO)-C-13, (CN-)-C-12, and (CN-)-C-13 on the IR spectrum of Hmd.