Methanobactin is an extracellular, copper-binding chromopeptide from the methane-oxidizing bacterium, Methylosinus trichosporium OB3b, believed to be involved in copper detoxification, sequestration, and uptake. Although small (1217.2 Da), methanobactin possesses a complex three-dimensional macrocyclic structure with several unusual moieties. The molecule binds one copper and has the N-2-isopropylester-(4-thionyl-5-hydroxyimidazolate)-Gly¹-Ser²-Cys³-Tyr⁴-pyrrolidine-(4-hydroxy-5-thionylimidazolate)-Ser⁵-Cys⁶-Met⁷ sequence [Kim, H. J., et al. (2004) Science 305, 1612−1615]. We report methods for purifying methanobactin from M. trichosporium OB3b and present initial evidence of its physiological function. MALDI-TOF MS was used to systematically monitor samples for optimizing purification conditions, and for detecting and analyzing specific metal−methanobactin complexes. Purification was performed by first stabilizing the extracted compound with copper followed by separation using reversed-phase HPLC in neutral pH buffers. Purified methanobactin exhibited UV−visible maxima at 342 nm, a shoulder at 388 nm, and a broad peak at 282 nm. These features were lost upon CuCl₂ titration with appearance of new features at 335, 356, 290, and 255 nm. Furthermore, methanobactin contains two fluorescent moieties, which exhibit broad emissions at 440−460 nm (λ_max^ex at 388 nm) and 390−430 nm (λ_max^ex = 342 nm), respectively. Finally, methanobactin eliminates the growth lag in M. trichosporium OB3b and substantially increases growth rates when cultures are exposed to elevated copper levels.