As a complex, cell-specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of L-arginine-nitric oxide (NO)-producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving L-arginine, a substrate of NO synthases (NOSs), or Nω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub-groups – acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. L-Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold-induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in L-arginine-treated rats. Ultrastructural examinations of L-arginine-treated cold-acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well-ordered cristae in their profiles), which however were inactive in L-arginine-receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, L-NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.