Bioactive SNOs are found in many tissues. We speculated SNOs might be misidentified in conventional assays which reduce NO⁻₃to NO.S-Nitrosothiols were exposed to saturated VCl₃in HCl, 1% KI in acetic acid, photolysis, or CuCl and CSH in He; NO was measured by chemiluminescence.S-Nitrosothiols were readily detected in VCl₃but not in KI. Reduction in CuCl/cysteine was linear (r²= 1.0,n= 6), sensitive to 10 pmol, and eliminated by HgCl₂; it did not detect NO⁻₂, NO⁻₃, or 3-nitrotyrosine.S-Nitrosothiols represented ∼2.9% of NO_xassayed by VCl₃in human serum, of which <5% were low-mass species. In summary, (i) conventional assays may misidentify NO⁻₃, but not NO⁻₂, as SNOs; and (ii) chemiluminescence/reduction systems may be sensitive and specific as SNO assays. We suggest that assay of the SNO fraction in biological NO_xmay be more relevant and feasible than is now appreciated.