Large conductance calcium activated (K_Ca) channels regulate the physiological functions of many tissues, including cerebrovascular smooth muscle. L-glutamic acid (glutamate) is the principal excitatory neurotransmitter in the central nervous system and oxygen tension is a dominant local regulator of vascular tone. In vivo, glutamate and hypoxia dilate newborn pig cerebral arterioles and both dilations are blocked by inhibition of CO production. CO dilates cerebral arterioles by activating K_Ca channels. Therefore, the present study was designed to investigate effects of glutamate and hypoxia on cerebral CO production and the role of K_Ca channels in the cerebral arteriolar dilations to glutamate and hypoxia. In the presence of iberiotoxin or paxillin that block dilation to the K_Ca channel opener, NS1619, neither CO nor glutamate dilated pial arterioles. Conversely, neither paxillin nor iberiotoxin inhibited dilation to acute severe or moderate prolonged hypoxia. Both glutamate and hypoxia increased CSF CO concentration. Iberiotoxin that blocked dilation to glutamate did not attenuate the increase in CSF CO. The guanylyl cyclase inhibitor, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one (ODQ), that blocked dilation to sodium nitroprusside did not inhibit dilation to hypoxia. These data suggest that dilation of newborn pig pial arterioles to glutamate is mediated by activation of K_Ca channels consistent with the intermediary signal being CO. Surprisingly, although heme oxygenase (HO) inhibition attenuates dilation to hypoxia, hypoxia increases CSF CO concentration, and K_Ca channel antagonists block dilation to CO, neither K_Ca channel blockers nor ODQ altered dilation to hypoxia suggesting the contribution of the HO/CO system to hypoxia-induced dilation is not by stimulating vascular smooth muscle K_Ca channels or guanylyl cyclase.