In addition to heme-irons, reactive (beta-globin thiols (betaCys93s) of hemoglobin (Hb) also have been shown to interact with endogenous nitric oxide (NO) thereby contributing to vascular tone regulation. What relative roles do these NO binding sites contribute to the overall Hb-mediated vasoactivity? Several test Hbs with either or both the NO binding sites preliganded or blocked were prepared and tested in a rat thoracic aortic ring model. Hbs tested were: NEM-Hb (ferrous Hb with masked thiols), HbNO (ferrous Hb preliganded with NO), Hb(+)CN (ferric Hb liganded with CN(-)), NEM-HbNO and NEM-Hb(+)CN (Hbs with both heme-iron and cysteine sites preliganded or blocked). Typically, >0.2 microM control Hb significantly increased isometric tension in agonist stimulated vessel rings (58.1 +/-7.0% over baseline). At comparable concentrations, NEM-Hb also caused a significant contraction (50.7+/-9.5%) while HbNO and Hb(+)CN did not (-5.5+/-6.0% and -3.7+/-4.6%, respectively). For these Hbs, masking thiols as well did not significantly alter respective vascular effects. Ferrous sperm whale myoglobin (Mb), which has no reactive thiol, elicited a significant contraction (55.1+/-13.2%) while metMb did not (-0.8+/-3.2%), suggesting the relative importance of heme-iron ligand and oxidation state in Hb vasoactivity. Additionally, ferrous or ferric equine heart cytochrome-C, a heme protein with no readily available heme-iron and cysteine binding sites, did not elicit notable contraction. Human Hb variants in which (betaCys93s are deleted or substituted with non-cysteine residues did not reveal any documented significant hemodynamic abnormalities. These results indicate that reactive globin-thiols do not appear to play a prominent role relative to heme-irons in Hb-mediated vasoconstriction.