Despite their ban several decades ago, polychlorinated biphenyls (PCBs) still pose a health threat to human beings due to their persistent and accumulative nature and continued presence in the environment. Non-dioxin-like (NDL)-PCBs have earlier been found to have effects on the immune system, including human neutrophil granulocytes. The aim of this study was to investigate the differences between ortho-chlorinated NDL-PCBs with a low or high degree of chlorination in their capability to induce the production of reactive oxygen species (ROS) in human neutrophil granulocytes in vitro. We used some of the congeners occurring at the highest levels in blood, breast milk and food: PCB 52 representing the low-chlorinated congeners and PCB 180 the high-chlorinated congeners. In addition, the extensively studied PCB 153 was included as a reference compound. ROS production was assessed with the luminol-amplified chemiluminescence and DCF fluorescence assays. The involvement of intracellular signalling mechanisms was investigated using different pharmacological substances. At high concentrations (10-20 μM), PCB 52 induced more ROS than PCB 153 and PCB 180. The role of extracellular signal-regulated kinase (ERK) 1/2 and/or ERK 5 signalling in PCB-induced ROS production was implicated through the reduction in ROS in the presence of the specific inhibitor U0126, whereas reduced ROS production after the use of SB203580 and SP600125 indicated the involvement of the p38 mitogen-activated protein kinase (MAPK) and c-Jun amino-terminal kinase (JNK) pathways, respectively. In addition, the calcineurin inhibitor FK-506, the intracellular calcium chelator BAPTA-AM and the antioxidant vitamin E reduced the levels of ROS. The intracellular signalling mechanisms involved in ROS production in human neutrophil granulocytes appeared to be similar for PCB 52, PCB 153 and PCB 180. Based on the results from the present and previous studies, we conclude that for abundant ortho-chlorinated PCBs found in the blood, low-chlorinated congeners induce higher production of ROS in neutrophil granulocytes than high-chlorinated congeners. This could be relevant during acute exposure scenarios when high concentrations of PCBs are present.