Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O(2)(-)), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O(2)(-) production in cultured human lymphoblast cell lines by three different methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using electron spin resonance (ESR) with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Lymphocytes were isolated and immortalized by an Epstein-Barr Virus (EBV)-transformation procedure. Superoxide was measured in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA). Cytochrome C and the spin traps EMPO and DEPMPO detected two to five times less superoxide compared to CMH. Thus, CMH provided the most quantitative measurement of superoxide generation in human lymphoblast cell lines. Superoxide detection with CMH was linear dependent on cell concentration and was inhibited by SOD but not by catalase. Both cell-permeable polyethylene glycol (PEG)-SOD and extracellular Cu,Zn-SOD inhibited O(2)(-) detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O(2)(-) generation in human lymphoblasts. Our study describes a new technique for O(2)(-) measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O(2)(-) in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.