Adenosine, astrocyte metabotropic glutamate receptors (mGluRs), and epoxyeicosatrienoic acids (EETs) have been implicated in neurovascular coupling. Although A(2A) and A(2B) receptors mediate cerebral vasodilation to adenosine, the role of each receptor in the cerebral blood flow (CBF) response to neural activation remains to be fully elucidated. In addition, adenosine can amplify astrocyte calcium, which may increase arachidonic acid metabolites such as EETs. The interaction of these pathways was investigated by determining if combined treatment with antagonists exerted an additive inhibitory effect on the CBF response. During whisker stimulation of anesthetized rats, the increase in cortical CBF was reduced by approximately half after individual administration of A(2B), mGluR and EET antagonists and EET synthesis inhibitors. Combining treatment of either a mGluR antagonist, an EET antagonist, or an EET synthesis inhibitor with an A(2B) receptor antagonist did not produce an additional decrement in the CBF response. Likewise, the CBF response also remained reduced by approximately 50% when an EET antagonist was combined with an mGluR antagonist or an mGluR antagonist plus an A(2B) receptor antagonist. In contrast, A(2A) and A(3) receptor antagonists had no effect on the CBF response to whisker stimulation. We conclude that (1) adenosine A(2B) receptors, rather than A(2A) or A(3) receptors, play a significant role in coupling cortical CBF to neuronal activity, and (2) the adenosine A(2B) receptor, mGluR, and EETs signaling pathways are not functionally additive, consistent with the possibility of astrocytic mGluR and adenosine A(2B) receptor linkage to the synthesis and release of vasodilatory EETs.