The reactivity of 4-nitrophenylpropionate has been examined in the presence of human milk lipase and cofactor solutions of the steroidal surfactants sodium-ursodeoxycholate, -glycoursodeoxycholate, -tauroursodeoxycholate, and -lithocholate, pregnanediol, and sodium fusidate and of the synthetic nonionic surfactant Antarox CO 730, in 0.01 mole dm −3 bistris propane buffer at pH 7.3, T = 310.5 K. The equilibrium surface tension of these cofactors has been measured under the same conditions but in the absence of substrate and enzyme and, in the cases where it was possible, critical micelle concentrations have been estimated. The steroidal molecules with a cis conformation and the nonionic synthetic surfactant stimulated the enzymatic activity, whereas the trans-steroid caused inhibition. The mechanism for reactivity has been discussed in terms of a stepwise association model for aggregation and the interactions between the surfactant, enzyme, and substrate.