The binding of ethanol and 1,1,1-trifluoroethanol (TFE) to both the H(mv) and H(ox) forms of soluble methane monooxygenase (sMMO) in solution has been studied by Q-band (35 GHz) CW and pulsed ENDOR spectroscopy of (1)H, (2)H and (19)F nuclei of exogenous ligands. As part of this investigation we introduce (19)F, in this case from bound TFE, as a new probe for the binding of small molecules to a metalloenzyme active site. The H(mv) form was prepared in solution by chemical reduction of H(ox). For study of H(ox) itself, frozen solutions were subjected to gamma-irradiation in the frozen solution state at 77 K, which affords an EPR-visible mixed-valent diiron center, denoted (H(ox))(mv), held in the geometry of the diiron(III) state. The (19)F and (2)H ENDOR spectra of bound TFE together with (1,2)H ENDOR spectra of bound ethanol indicate that the alcohols bind close to the Fe(II) ion of the mixed-valence cluster in H(mv) and in a bridging or semi-bridging fashion to H(ox). DMSO does not affect the binding of either of the ethanols or of methanol to H(ox), nor of ethanol or methanol to H(mv). It does, however, displace TFE from the diiron site in H(mv). These results provide the first evidence that crystal structures of sMMO hydroxylase into which product alcohols were introduced by diffusion represent the structures in solution.