The polarity of water solubilized in the interior of non-ionic surfactant reverse micellar systems has been investigated using optical probes. Small amounts of water incorporated on the inside of various reverse micellar systems, polyoxyethylene sorbitan trioleate (Tween-85)/isopropyl alcohol/n-hexane mixture and polyoxyethylene tert-octylphenyl ether (TX-100)/cyclohexane, were found to have low polarity compared with bulk water. The polarity of water in both Tween-85 and TX-100 reverse micellar systems corresponded to that of methanol. The activity of the proteases alpha-chymotrypsin and subtilisin in Tween-85 and TX-100 reverse micellar systems has also been investigated. It was found that the amounts of water in the reverse micellar system have a significant influence on the activity of enzymes. In the TX-100 reverse micellar system, neither enzymes showed any activity. In contrast, the activity of enzymes in the Tween-85 reverse micellar system was similar to that in the aqueous system and followed standard Michaelis-Menten kinetics. (C) 2004 Society of Chemical Industry.