Lucigenin-enhanced chemiluminescence (LECL) is widely used for the detection of reactive oxygen species released from various cells and mitochondria. However, the LECL response varies depending on cell species and assay conditions at least in part by unknown factors. Here we report that cell adhesion is an important factor for increasing LECL of tetradecanoylphorbol acetate (TPA)-stimulated human neutrophils. More than 90% LECL remained even after complete removal of the cell suspension 10 min after TPA stimulation, and approximately 22.5% of neutrophils were adhered to the reaction tube. These results indicate that LECL by an adhering neutrophil is approximately 45x higher than that by a non-adhering neutrophil. LECL by leukocyte adhesion deficiency neutrophils was one-fifth of that by normal neutrophils and completely disappeared when the cell suspension was removed, confirming that LECL depends highly on cell adhesion. The oxidase activity of adhering neutrophils measured after permeabilization with Renex 30 together with NADPH addition was similar to that of non-adhering neutrophils, indicating that lucigenin and cell adhesion do not enhance the oxidase activity. Based on these findings, we propose that a mixture of adhering and non-adhering neutrophils can be used for simultaneous screenings of adhering activity and the oxidase activity of neutrophils.