Cerebrovascular CO₂ reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated dilatation (cSMD) that may better reflect endothelial function. We aimed to determine the nitric oxide (NO)-dependency of CVR and cSMD. Eleven volunteers underwent a steady-state CVR test and transient CO₂ test of cSMD during intravenous infusion of the NO synthase inhibitor N^G-monomethyl-l-arginine (l-NMMA) or volume-matched saline (placebo; single-blinded and counter-balanced). We measured cerebral blood flow (CBF; duplex ultrasound), intra-arterial blood pressure and (Formula presented.). Paired arterial and jugular venous blood sampling allowed for the determination of trans-cerebral NO₂⁻ exchange (ozone-based chemiluminescence). l-NMMA reduced arterial NO₂⁻ by ∼25% versus saline (74.3 ± 39.9 vs. 98.1 ± 34.2 nM; P = 0.03). The steady-state CVR (20.1 ± 11.6 nM/min at baseline vs. 3.2 ± 16.7 nM/min at +9 mmHg (Formula presented.); P = 0.017) and transient cSMD tests (3.4 ± 5.9 nM/min at baseline vs. −1.8 ± 8.2 nM/min at 120 s post-CO₂; P = 0.044) shifted trans-cerebral NO₂⁻ exchange towards a greater net release (a negative value indicates release). Although this trans-cerebral NO₂⁻ release was abolished by l-NMMA, CVR did not differ between the saline and l-NMMA trials (57.2 ± 14.6 vs. 54.1 ± 12.1 ml/min/mmHg; P = 0.49), nor did l-NMMA impact peak internal carotid artery dilatation during the steady-state CVR test (6.2 ± 4.5 vs. 6.2 ± 5.0% dilatation; P = 0.960). However, l-NMMA reduced cSMD by ∼37% compared to saline (2.91 ± 1.38 vs. 4.65 ± 2.50%; P = 0.009). Our findings indicate that NO is not an obligatory regulator of steady-state CVR. Further, our novel transient CO₂ test of cSMD is largely NO-dependent and provides an in vivo bioassay of NO-mediated cerebrovascular function in humans. (Figure presented.). Key points: Emerging evidence indicates that a transient CO₂ stimulus elicits shear-mediated dilatation of the internal carotid artery, termed cerebral shear-mediated dilatation. Whether or not cerebrovascular reactivity to a steady-state CO₂ stimulus is NO-dependent remains unclear in humans. During both a steady-state cerebrovascular reactivity test and a transient CO₂ test of cerebral shear-mediated dilatation, trans-cerebral nitrite exchange shifted towards a net release indicating cerebrovascular NO production; this response was not evident following intravenous infusion of the non-selective NO synthase inhibitor N^G-monomethyl-l-arginine. NO synthase blockade did not alter cerebrovascular reactivity in the steady-state CO₂ test; however, cerebral shear-mediated dilatation following a transient CO₂ stimulus was reduced by ∼37% following intravenous infusion of N^G-monomethyl-l-arginine. NO is not obligatory for cerebrovascular reactivity to CO₂, but is a key contributor to cerebral shear-mediated dilatation.