The influence of the monovalent cations on the UV-visible spectra of the methylamine dehydrogenase (MADH) from bacterium W3A1 was investigated. The spectra for the oxidized and 1- and 2-electron-reduced forms, unperturbed by bound cations, were obtained for the enzyme, and the extinction coefficients for these forms were determined. The binding of the following cations was investigated: Li+, Na+, K+, Rb+, Cs+, NH4+, (CH3)3NH+, and (CH3)4N+. It was shown that each cation produced unique spectral changes, some of which were pH-dependent. Except for NH4+, all spectral changes produced by binding of the monovalent cations can be explained by assuming two different binding sites in MADH (type I and type II sites). Na+ and K+ displayed monophasic binding to the type II site, (CH3)3NH+ and (CH3)4N+ displayed monophasic binding to the type I site, and Li+, Rb+, and Cs+ displayed either monophasic or biphasic binding to one or both sites depending on pH. The pH dependence for binding to the two sites is different, i.e. plots of log(Kd) versus pH have negative slopes approximately 1 for the type II site, whereas the negative slope is significantly less than 1 (0.6-0.8) for the type I site. This difference leads to pH-dependent changes in spectral features produced by binding of Li+, Rb+, and Cs+. The spectral changes seen during titrations with NH+4 were unlike those seen for any other cation. The binding of NH+4 was biphasic, and the spectra produced in each phase were unaffected by pH. It is assumed that this cation binds to the tryptophan tryptophylquinone cofactor to produce the iminoquinone in the first phase and then binds to the type I monovalent cation binding site in the second phase. It is suggested that binding of NH+4 (and CH3NH+3) to the type I site is a prelude to binding to the cofactor.