Theophylline is an effective bronchodilatator used in the treatment of asthma which requires frequent control because of its narrow therapeutic index. Over the past decade much attention has been dedicated to the peculiar properties of the inner water pools of AOT (sodium 2-bishexyl-ethyl sulfosuccinate) microemulsions as enzyme microreactors, yet few analytical applications of the latter have been reported. We developed an original assay based on the uncompetitive inhibition by theophylline of the reaction catalyzed by alkaline phosphatase from bovine liver (E.C. 3.1.3.1) of the ELF-97 fluorogenic substrate in borate buffer 20 mM (pH 8.6)/AOT/iso-octane-ethyl acetate (95:5) at a temperature of 37 degrees C. Optimal activity of endogenous plasmatic alkaline phosphatase isoenzymes approximately pH 10.5, interfering activity of the serum are avoided. The assay is multiple point rate, monitoring the appearance of the photostable fluorescence emission of the reaction product (510-530 nm) out of the water pool. The influence of several parameters such as the amount of buffer (W(o)), the amount of alkaline phosphatase, sample volume (10-30 microl) [corrected], optimal run time (1-7 min) and the use of phosphorylating acceptor (2A2MP) are discussed. The method was compared to HPLC UV and TDx methods.