A water-soluble, chemiluminescent substrate for sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) activity was synthesized. This was achieved by replacing the diacylglycerol moiety found in the natural substrate, phosphatidylinositol, by a dioxetane-containing moiety, resulting in the synthetic substrate (+/-)-3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo[3.3.1.1(3,7]decan]-4-yl)phenyl myo-inositol-1-O-hydrogen phosphate (LUMI-PI). PI-PLC-catalyzed cleavage of the phosphodiester bond of LUMI-PI results in the formation of a chemiluminescent precursor, the labile anion AMP-D, which decays with emission of light. An assay procedure was developed using 96-well microtiter plates with detection of the chemiluminescence on autoradiography film. With this procedure the detection limit of purified phospholipase C was about 10 pg. Furthermore, PI-PLC activity was readily detected in situ using small quantities (< 1 microliters) of liquid cultures of PI-PLC-producing bacterial strains.