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In vivo studies show a dynamic cycle in which α-nitrosylated hemoglobin is mainly in the relaxed state in arterial blood of rats treated with 2-(N,N-diethylamino)-diazenolate-2-oxide, but converts mainly to the tense state during the arterial-venous transit. A detailed analysis shows that different electron paramagnetic resonance spectra recorded for α-nitrosyl hemoglobin in arterial and venous blood at 77 K originate only from a different ratio between 5- and 6-coordinate heme without any change in the concentration of nitrosyl hemoglobin. In venous blood, the five- and six-coordination equilibrium of the α-nitrosyl heme is shifted in favor of the 5-coordinate state (58% venous vs. 20% arterial). These results are not consistent with the recently proposed exchange of nitrosyl heme with the β-93 nitrosothiol group of hemoglobin during the arterial-venous cycle. [Copyright &y& Elsevier]