Plasma levels of nitrite/nitrate may not accurately reflect endothelial nitric oxide synthase (eNOS) function because of interference by dietary nitrates. Nitrosyl hemoglobin (HbNO), a metabolic product of nitric oxide (NO*), may better correlate with bioavailable NO*, but it may depend on the activity of different NOS isoforms and may be affected by dietary nitrite/nitrate. This work examined the correlation between vascular endothelial NO* release and blood levels of HbNO. We measured HbNO in mouse blood using electron spin resonance (ESR) spectrometry, and we quantified vascular production of NO* using colloid Fe(DETC)2 and ESR. C57Blk/6 mice who were fed a high-nitrate diet had levels of plasma HbNO increased 10-fold, whereas those fed a low-nitrate diet had decreased HbNO levels from 0.58 +/- 0.02 to 0.48 +/- 0.01 microM. Therefore, a low-nitrate diet is essential when using HbNO as a marker of eNOS activity. Treatment with L-NAME and the eNOS-specific inhibitor L-NIO halved HbNO formation, which reflects the complete inhibition of NO* release by aorta endothelium. Treatment of mice with the selective inducible NOS (iNOS) inhibitor, 1400W, or the selective neuronal NOS (nNOS) inhibitor N-AANG did not alter either blood HbNO levels or vascular NO*. The relationship between HbNO and NO* production by the endothelium (0.23 microM HbNO to 5.27 microM/h of NO*/mg of dry weight aorta) was found to be identical for both C57Blk/6 mice and mice with vascular smooth muscle-targeted expression of p22phox associated with strong increase in eNOS activity. These results support the important role of eNOS in the formation of circulating HbNO, whereas iNOS and nNOS do not contribute to HbNO formation under normal conditions. These data suggest that HbNO can be used as a noninvasive marker of endothelial NO* production in vivo.