The observation that aliphatic diamines become poor substrates as the carbon chain length decreases and that ethylenediamine, the shortest diamine, is an irreversible inhibitor of lysyl oxidase led to the investigation of the mechanism of inhibition by ethylenediamine. The cis but not the trans isomer of 1,2-diaminocyclohexane was also a potent irreversible inhibitor of lysyl oxidase, consistent with the interaction of both amino groups of vicinal diamines with an enzyme moiety. Both cis-1,2-diaminocyclohexane and ethylenediamine but not trans-1,2-diaminocyclohexane markedly perturbed the spectrum of free pyrroloquinoline quinone (PQQ), a covalently linked form of which is the carbonyl cofactor of lysyl oxidase. cis-1,2-Diaminocyclohexane also induced similar changes in the spectrum of lysyl oxidase. The perturbations of the spectra of PQQ or of lysyl oxidase by cis-1,2-diaminocyclohexane or ethylenediamine as well as the development of irreversible inhibition of the enzyme by cis-1,2-diaminocyclohexane or ethylenediamine were all markedly reduced under anaerobic conditions. Moreover, approximately 1 mol of H2O2 was released per mol of PQQ or lysyl oxidase upon aerobic incubation with cis-1,2-diaminocyclohexane, while approximately 2 mol of 3H+ were released from cis-[1,2-3H] 1,2-diaminocyclohexane per mol of PQQ or lysyl oxidase under corresponding conditions. A proposal for the mechanism of inhibition of lysyl oxidase by vicinal diamines is presented which involves limited oxidation of the diamine linked to PQQ at the active site so that the PQQ-diamine complex is finally stabilized by a conjugated 6-membered ring.