This study examined the role of calcium in the generation of reactive oxygen species (ROS) in human lymphocytes activated by the chemoattractant formyl-Met-Leu-Phe (fMLP) and the T-cell mitogen phytohaemagglutinin (PHA). The concentrations of cytosolic calcium ([Ca2+]i) and ROS were monitored simultaneously with a fluorescence spectrophotometer after the cells had been incubated in fura-2 (calcium-sensitive dye) and 2',7'-dichlorofluorescein diacetate (DCF-DA, ROS-sensitive dye). The lymphocytes were stimulated with fMLP (200 nmol l-1) or PHA (10 µmol l-1) in the absence and presence of extracellular calcium. A dose-response test was also conducted for extracellular calcium. fMLP and PHA significantly increased both [Ca2+]i (P < 0.001) and ROS concentrations (P < 0.001) above the control levels in the presence of extracellular calcium. However, such increases were abolished in the absence of extracellular calcium, suggesting total dependence of the responses to both fMLP and PHA on transplasma-membrane calcium influx. There were also graded increases in ROS with increasing concentrations of extracellular calcium. The results show that transplasma-membrane calcium influx is essential for fMLP- and PHA-induced generation of reactive oxygen species in human lymphocytes.