This work describes a technique for the rapid and sensitive determination of acetylcholine, urea, and penicillin in flowing solution streams using stabilized systems of solventless bilayer lipid membranes (BLMs). This method of monitoring substrates of hydrolytic enzyme reactions made use of BLMs which were supported on ultrafiltration membranes such as polycarbonate and glass microfiber; these filter membranes were found to enhance the stability of BLMs for uses in flow injection experiments. The enzymes were immobilized on BLMs by incorporating the protein solution into the lipid matrix at the air/electrolyte interface before the BLM formation, followed by injections of the substrates into flowing streams of a carrier electrolyte solution. Hydronium ions produced by the enzymatic reaction at the BLM surface caused dynamic alterations of the electrostatic fields and phase structure of BLMs, and as a result ion current transients were obtained; the magnitude of these signals was correlated to the substrate concentration, which could be determined at the micromolar level. The response times were ca. 10 s, and acetylcholine, urea, and penicillin could be determined in continuous flowing systems with a maximum rate of 220 samples/h. It is expected that this analytical utility of stabilized BLMs for flow stream uses will provide new opportunities in this strategy of chemical sensing.