The biological roles of nitric oxide (NO)-hemoglobin (Hb) derivatives are obscure. It is proposed that NO can function as an allosteric regulator of hemoglobin oxygen-binding properties. We aimed to estimate the effects of NO donors and NO-synthase substrate (L-arginine) on hemoglobin-oxygen affinity (HOA) in experiments in vitro with the various ratios between NO formed and Hb and various oxygen pressures. HOA index (p50), blood pH, plasma and red blood cell (RBC) concentrations of nitrite/nitrate and methemoglobin amounts were measured after the experiments. In our experiments, blood incubation with NO donors (glyceryltrinitrate, molsidomine, sodium nitroprusside, S-nitrosocysteine) or NO-synthase substrate (L-arginine) did not change HOA even at NO:Hb ratio of 1:1. At the same time our results showed that oxygenated blood incubation with S-nitrosocysteine induced an oxyhemoglobin dissociation curve shift leftwards. This indicates a leading role of met-Hb in a modification of Hb oxygen-binding properties. However other NO-modified forms of hemoglobin (S-nitroso- and nitrosylhemoglobin) also may be involved in the regulation of HOA. The results obtained indicate that nitric oxide can be the allosteric effector of hemoglobin, increasing or decreasing its oxygen affinity - possibly, through the generation of different NO-Hb derivatives.