In this article, the extraction of cytochrome c utilizing various nonionic surfactant microemulsions has been tested to determine the effect of surfactant structure on protein partitioning. Surfactants tested include a linear alcohol ethoxylate (Neodol 91-2.5), two alkyl phenol ethoxylates (lgepal CO-520, Trycol 6985), and a series of alkyl sorbitan esters that are either ethoxylated (Tweens) or un-ethoxylated (Spans). Initial attempts to extract hemoglobin into Neodol 91-2.5 Winsor II microemulsions (oil-continuous) appeared successful based on heme estimation. Careful analysis showed that the hemoglobin had dissociated prior to extraction and that only the heme was extracted with false positive results. In fact, Neodol 91-2.5 microemulsions were unable to extract a variety of proteins with differing biophysical properties. Among all the other nonionic surfactant microemulsions tested only those made using sorbitan esters extracted significant amounts of cytochrome c. The partition coefficients achieved in this study are more than an order of magnitude higher than that seen previously in the literature for comparable sorbitan systems. However, this partition coefficient is extremely sensitive to ionic strength. At an ionic strength as low as 0.001 M, the partition coefficient is reduced to that seen in previous studies. We have found that protein partitioning in sorbitan ester microemulsions is not a function of water content. In addition, extraction is not a function of either alkyl chain length, or polyethylene oxide molecular weight. Hence, the sorbitan group appears to have an important role in extraction, possibly through a weak electrostatic protein-surfactant interaction. (c) 1995 John Wiley & Sons, Inc.