Abstract: Eighteen kinds of pyrimido[5,4-d]pyrimidines together with several commercially available fluorescent compounds such as perylene, Rhodamine B, etc., were evaluated as the reagents for a peroxyoxalate chemiluminescence (CL) detection system by using a flow injection method. The peroxyoxalate CL reaction employed consists of bis(2,4,6-trichlorophenyl)oxalate, hydrogen peroxide, triethylamine, and a fluorophore. Under the conditions used, 2,6-bis[di-(2-hydroxyethyl)amino]-4,8- dipiperidinopyrimido[5,4-d]pyrimidine (Dipyridamole) and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine (1i) gave very intense chemiluminescence intensities which were larger than those of any other commercially available fluorescent compounds tested (e.g., 10 times larger than that of perylene). Derivatives of pyrimido[5,4-d]pyrimidine in CHCl3 or methanol were injected into a stream of 3 mM triethylamine in acetonitrile (1.0 mL min-1), which was combined with a stream of 0.15 mM bis(2,4,6-trichlorophenyl)oxalate and 4.5 mM H2O2 in acetonitrile (1.0 mL min-1) and the intensity of the chemiluminescence generated was measured. Standard curves were prepared with use of known concentration. of each fluorescent compound. Results are tabulated for 17 compounds. Very intense chemiluminescence was observed with use of dipyridamole and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine.