Nitric oxide synthases (NOSs) have been shown to participate in the mechanism of the antinociceptive action of tapentadol. The results obtained in this study indicate that tapentadol administered intrathecally at a range of doses (30-100 µg) increased nociceptive thresholds in the Randall-Selitto and tail-flick tests in rats; however, this effect was significant only for the higher doses. After intracerebroventricular administration of tapentadol at the same dose range, an antinociceptive effect was observed only in response to mechanical stimuli. In coadministration studies, L-N-nitro arginine (L-NOArg) - a nonselective NOS inhibitor as well as selective inhibitors: 7-Nitroindazole (7-NI), L-N(1-iminoethyl)lysine (L-NIL) or N-(1-iminoethyl)-L-ornithine (L-NIO) for the respective neuronal, inducible, and endothelial NOSs enhanced the antinociceptive activity of intrathecally administered tapentadol in the Randall-Selitto test and to a lesser extent in the tail-flick test. A similar, although less pronounced effect of intracerebroventricular tapentadol was also observed after previous administration of NOS inhibitors in the Randall-Selitto test, but not in the tail-flick test. In conclusion, neuronal NOS, inducible NOS, and endothelial NOS influence the antinociceptive action of tapentadol at the spinal level and to a much lesser extent at the supraspinal level.