Lipid oxidation generates secondary oxidation products, which compromise myoglobin (Mb) redox stability. Although lipid oxidation-induced discoloration in carboxymyoglobin (COMb) is documented, the molecular basis for interactions between COMb and lipid oxidation products has not been investigated. Our objective was to characterize the adduction of 4-hydroxy-2-nonenal (HNE), a reactive lipid oxidation product, with COMb, utilizing mass spectrometry. Equine COMb and equine OxyMb (0.15mM) were incubated with HNE (1.0mM) at pH 7.4, 37°C (physiological condition) for 6h, and at pH 5.6, 4°C (typical meat storage condition) for 7days. The samples were analyzed in Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). MS spectra revealed that HNE formed mono-, di-, and tri-adducts with COMb at physiological conditions, whereas mono-, di-, tri-, and tetra-adducts were detected in OxyMb. This observation suggested a lower reactivity of COMb towards HNE at physiological conditions, compared to OxyMb. In contrast, at meat storage conditions, HNE formed mono- and di-adducts with both COMb and OxyMb, thus revealing a similar trend for aldehyde adduction in the cherry-red colored Mb redox forms. The present study is the first to report HNE adduction in COMb, and proteomic investigations are underway to determine the sites of HNE adduction in COMb.