Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5 x 10(-7) M and 1 x 10(-6) M respectively with r.s.d. < 2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months.