The light-emitting species of chemiluminescence produced in rat liver homogenate on adding autoxidized linseed oil (AOLO) were investigated. The chemiluminescent intensity of liver homogenate was strongly enhanced by the addition of AOLO and showed a proportional relationship to the amount of AOLO. The chemiluminescence was reduced with singlet oxygen (102) quenchers and free radical scavengers. Among them, β-carotene showed the most effective quenching. The emission spectrum had broad bands in the visible region with eminent chemiluminescent lines at 520, 575 and 640 nm due to the simultaneous transition, 2[1Δg]→2[3Σg -]. An additional weak line was found at 480 nm corresponding to [1Δg] + [1Σg +]→[3Σg -]. In the presence of β-carotene, lines corresponding to the simultaneous transition of 1O2 disappeared. These results indicate that the liver homogenate with AOLO generated singlet molecular oxygen as one of the major light-emitters of the chemiluminescence. A possible mechanism for the generation of 1O2 is by decomposition of peroxy radicals derived from AOLO in the liver homogenate.