Nitric oxide (NO) is formed in gastrointestinal tract by both intestinal mucosa and commensal microbiota. In this work, NO production by probiotic bacteria Lactobacillus plantarum 8PA3 was demonstrated by electron paramagnetic resonance (EPR)and NOspecificfluorescent dyes DAF-FM DA (4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate) and DAA (1,2-diaminoanthraquinone sulfate). After we ruled out the generation of NO via denitrification, we showed that NO production by L. plantarumdepends on L-arginine what is typical for NO-synthases (NOS), and thus proposed NOS-activity in L. plantarum. However,we were unable to inhibit the NO production with three specific NOS inhibitorsL-NAME, L-NIL, and nNOS Inhibitor I.We also demonstrated an increased level of NO production in lactobacilli with damaged cellular membranes suggesting stress-dependent NO production by L. plantarum.