A careful study of the parameters affecting the chemiluminescent reaction of TCPO (bis(2,4,6-trichlorophenyl)oxalate) and 2-NPO (bis(2-nitrophenyl)oxalate) with hydrogen peroxide, in the presence of imidazole (used as buffer and catalyst) and the fluorophore 3-AFA (3-aminofluoranthene), was carried out in acetonitrile/water medium. The data are reported in terms of the time (tImax) required for the relative maximum chemiluminescence intensity (Imax) and the area (A) under the curve of intensity νs. time. At controlled acidity and high unprotonated imidazole concentration ([IMI] = 1x10−2mol.L−1), the Imaxand tImaxvalues do not depend on the acetonitrile/water ratio. The CL intensity is maximum at around pH 6.0 (higher buffer capacity). For 1x10−2< [IMI] < 1x10−4mol.L−1, the area is largest when [IMI] = 1x10−3mol.L−1and linearly dependent on the hydrogen peroxide concentration (10−5− 10−3mol.L−1). The area is also enhanced by increasing the fluorophore concentration. The acidity controls the unprotonated imidazole and H+concentration. Several errors can arise from quantitative analyses carried out without control of the HIMI+and IMI concentrations.