Multidrug resistance protein 5 (MRP5) has been linked to cGMP cellular export in peripheral vascular smooth muscle cells (VSMCs), and is widely expressed in brain vascular tissue. In the present study, we examined whether knockdown of MRP5 in pial arterioles (via antisense oligodeoxynucleotide [ODN] applications) affected NO/cGMP-induced dilations. The antisense, or (as a control) missense ODN, was applied to the cortical surface ~18h prior to study via closed cranial windows. The efficacy of the antisense vs missense ODN in eliciting selective reductions in MRP5 expression was confirmed by analysis of MRP5 mRNA in pial tissue. Unexpectedly, in initial studies, a significantly lower maximal pial arteriolar diameter increase in the presence of the NO donor, SNAP, was seen in the antisense vs missense ODN-treated rats (35 vs 48% diameter increase, respectively). It was suspected that this related to a reduced VSMC sensitivity to cGMP due to prolonged exposure to increased intracellular cGMP levels elevated by overnight restriction of cGMP efflux. That postulate was supported by a finding of a diminished vasodilating response to the PKG-activating cGMP analogue, 8-pCPT-cGMP, in antisense vs missense ODN-treated rats. To prevent desensitization, additional rats were studied in the presence of chronic NOS inhibition via L-NNA. In the NOS-inhibited rats, the maximal SNAP response was much higher in the antisense (62% increase) vs the missense ODN (40% increase) group. A similar result was obtained when monitoring responses to the sGC-activating drugs, YC-1 and BAY 41-2272. Moreover, in the presence of NOS inhibition, the normal SNAP-induced rise in periarachnoid CSF cGMP levels, which reflects cGMP efflux, was absent in the antisense ODN-treated, rats-a finding consistent with loss of MRP5 function. In conclusion, if one minimizes the confounding effects of basal cGMP production, a clearer picture emerges-one that indicates an important role for MRP5-mediated cGMP efflux in the regulation of NO-induced cerebral arteriolar relaxation.