A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X-45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X-45 less than 0.6, thermodynamically stable reverse micelles of water content (W-0) up to 30 are formed. Di(2-ethylhexyl) phosphoric acid (HDEHP; 1-2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine-rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper-chelate reverse micelles (Cu-RM) are characterized for their W-0, hydrodynamic radius (R-h), and aggregation number (N-ag). Similar with reverse micelles of bis-2-ethylhexyl sodium sulfosuccinate (AOT), R-h of the Cu-RM is also linearly related to W-0. However, N-ag is determined to be 30-90 at W-0 of 5-30, only quarter to half of the AOT reverse micelles. Then, selective metal-chelate extraction of histidine-rich protein (myoglobin) by the Cu-RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine-tagged proteins expressed as inclusion bodies. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 150-158, 2010