Information about the catalytic and kinetic properties of mitochondria NO-synthase from uterus smooth muscle is missing currently. According to the data on MitoTracker Orange CM-H2TMRos and 4-аmino-5-methylamino-2',7'-difluorescein, diaminofluorescein-FM (DAF-FM) dye co-localization in uterine smooth muscle cells, presented in this paper, NO can be synthesized in their mitochondria. High activity of NO synthase requires the presence of substrates of respiration, L-arginine, Ca 2+ and NADPH. It is established that the dependence of NO production on the concentration of L-arginine has a bell-shaped character with a maximum of 75 μM, and the apparent affinity constant for L-arginine is 28.9 ± 9.1 μM. The dependence of NO production on Ca 2+ concentration has a maximum at 100-250 μM; the activation constant for Ca 2+ is 44.4 ± 14.5 μM. The inhibitor of Ca 2+ transport in mitochondria ruthenium red (RuR), as well as the inhibitor of NO-synthase N G -nitro-L-arginine (NA), reduces NO production. The biosynthesis of NO by mitochondria depends on its energized level: it is stimulated by the addition of respiration substrates, suppressed with specific inhibitors of the electron transport chain (rotenone and antimycin A) and carbonyl-cyanide 3-chlorophenylhydrazone (CCCP) protonophore.