Although the phenomenon of opioid tolerance has been widely investigated, neither opioid nor nonopioid mechanisms are completely understood. The aim of the present study was to investigate the role of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the development of morphine-induced analgesia tolerance. The study was carried out on male Wistar albino rats (weighing 180-210 g; n = 126). To develop morphine tolerance, animals were given morphine (50 mg/kg; s.c.) once daily for 3 days. After the last dose of morphine was injected on day 4, morphine tolerance was evaluated. The analgesic effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), BAY 41-2272, S-nitroso-N-acetylpenicillamine (SNAP), N(G)-nitro-L-arginine methyl ester (L-NAME), and morphine were considered at 15 or 30 min intervals (0, 15, 30, 60, 90, and 120 min) by tail-flick and hot-plate analgesia tests (n = 6 in each study group). The results showed that YC-1 and BAY 41-2272, a NO-independent activator of soluble guanylate cyclase (sGC), significantly increased the development and expression of morphine tolerance, and L-NAME, a NO synthase (NOS) inhibitor, significantly decreased the development of morphine tolerance. In conclusion, these data demonstrate that the nitric oxide-cGMP signal pathway plays a pivotal role in developing tolerance to the analgesic effect of morphine.