Protein extraction into Tween 85 nonionic microemulsions is characterized. Apparently, a weak electrostatic interaction is the underlying extraction mechanism. The extraction of several proteins is examined as a function of protein size and net charge. The proteins that extracted successfully are positively charged. A size-exclusion limit (60 kDa) is also evident in these extractions. Tween 85 and several other nonionic surfactants possess a net negative charge at neutral pH. Titration measurements are an effective means of quantifying charge on these surfactants. In Tween 85 microemulsion separations, both the sign and the magnitude of surfactant charge play a role in extraction. Blends of Neodol 91-2.5 (capable of extracting 0% protein) and Tween 85 (capable of extracting 100% protein) indicate that a minimum net surfactant charge of 15 mequiv/mol is required for extraction.