PURPOSE: To determine an enzyme which is present alone or in combination with other substance in a sample by chemiluminescence reaction by using a stable chemiluminescent dioxetane which is induced by the enzyme to generate chemiluminescence.

CONSTITUTION: (A) An enzyme which is present alone or in combination with other substance in a sample is reacted with a stable 1,2-dioxetane of formula I [R1, R3 and R4 are each a passive organic group permitting occurrence of light; R2 is an aryl substituted with OX; X is a group readily reacting with an enzyme] and the enzyme is determined by the chemiluminescence reaction. Furthermore, 1,2-dioxetane is passed through 1,2-dioxetane compound which is an unstable oxide intermediate and decomposed into two carbonyl-containing compounds of formula II and formula III and simultaneously generates light by removing X from the 1,2-dioxetane.