The reversed micellar solution formed by Sorbitan trioleate (Span 85) coupled with Cibacron Blue F3G-A (CB) as an affinity ligand show good potential for use in reversed micellar protein extraction operations. In this work, the extraction behavior of lysozyme and ovalbumin with the reversed micellar phase has been investigated at extensive pH values and CB concentrations. The partitioning isotherms of lysozyme and ovalbumin were expressed by the Langmuir equation. By addition of 3 vol.% hexanol to the reversed micellar phase, the extraction capacity of lysozyme reached 6.38 mg/mL, over seven times higher than that of ovalbumin (0.83 mg/mL). Thus, lysozyme was separated from crude chicken ovalbumin solutions with a purification factor over 20. Moreover, the reversed micellar solution was recycled three times for the separation, and the purification factor was kept nearly 20 in the recycling. Incomplete back extraction of lysozyme to the stripping solution compromised the total recovery yields in the following recycles, so more effective back extraction method needs to be developed to make the extraction system more attractive. In general, the present results have shown the potential of the affinity-based reversed micellar system for application in protein separations. (c) 2006 Elsevier B.V. All rights reserved.