Assessment of leukocyte activity as the main source of ROS in seminal fluid of patients with normospermia and pathospermia using an original protocol based on the kinetic chemiluminescence method and adapted for semen analysis. A prospective study was attended by 95 men of reproductive age who applied to the Research Center of Medical Genetics (Moscow) for semen analysis. The material for the study were samples of native ejaculate. Chemiluminescent measurements were performed on a Lum-1200 chemiluminometer (DISoft, Russia) using the original method. Both in amplitude of basal and stimulated response, between the "normozoospermia", "pathozoospermia" and "pathozoospermia + leucospermia" groups, significant differences were obtained in the level of ROS production by leukocytes (p<0.05): the median level of basal chemiluminescence, normalized on the count of leukocytes was 0.13, 0.71 and 1.78, respectively; the median level of stimulated chemiluminescence normalized to the number of leukocytes was 0.62, 2.14, 5.94, respectively. The level of stimulated response did not exceed 0.5 arb. units in normozoospermic samples. In pathospermic groups, the level of stimulated response was low in about a third of semen samples, it was moderate in one third of patients, and high in one third of patients. A protocol previously developed for blood analysis was adapted to analyze the total ROS level produced by leukocytes in seminal fluid. In the groups of "pathozoospermia", "pathozoospermia + leucospermia", the level of basal ROS production by leukocytes was about 5 and 15 times higher than in the "normozoospermia" group, the level of stimulated ROS production was 3.5 and 9.5 times; this indicates oxidative stress, including with a normal number of leukocytes.