Recent observations suggested that nitric oxide (NO) has a role in triggering the early endothelial dysfunction in Kawasaki disease (KD). We investigated the amount of NO in conjunction with reactive oxygen species (ROS) produced by neutrophils in children with acute KD by a newly developed flow cytometric analysis. Forty children with acute KD (n = 14), non-KD febrile disease (n = 14), and afebrile control (n = 12) were enrolled (age, 3 to 88 months). Neutrophils in KD produced significantly higher amount of NO compared to others (p < 0.05). With regard to ROS, significant increase was not only found in KD but also in non-KD febrile children (p < 0.05 and p < 0.01, respectively). In KD patients, the amount of NO produced by neutrophils decreased after immunoglobulin (IVIG) treatment, while there was no significant change in ROS production. The amount of NO in KD patients also correlated well with the days from the onset. One patient who developed coronary arterial lesion showed the highest value of NO. In conclusion, neutrophils in acute KD generate both NO and ROS considerably, while NO production is exclusive in the early stage of KD before IVIG treatment. Abnormal immune system in KD might be characterized by an overproduction of NO, whereas the role of NO in endothelial damage remains to be elucidated.