We examined whether the glia limitans (GL) influences pial arteriolar relaxation elicited in vivo by the purinergic (P(2)Y(1) receptor) agonist ADP in female rats, and whether that influence is altered in ovariectomized (Ovx) females. A validated model for GL injury was used, topical application of the gliotoxin L-alpha-aminoadipic acid (L-alphaAAA), 24 h before the study. In both intact and Ovx females, L-alphaAAA had no effect on responses to the NO donor, S-nitroso-N-acetyl penicillamine, but ADP-induced pial arteriolar dilations were significantly reduced (by 33-90%), compared with vehicle-treated controls. When N(G)-nitro-L-arginine (L-NNA) was administered to L-alphaAAA-treated rats, the ADP response was virtually lost in intact females, but no further reductions were observed in the Ovx rats. On the other hand, in L-alphaAAA-treated Ovx females, when the gap junction blocker, Gap 27, was subsequently added to the suffusate, ADP reactivity fell to very low levels. In vehicle-treated control rats, L-NNA and Gap 27 reduced ADP reactivity by approximately 50% in intact and Ovx females, respectively. An earlier study indicated that the endothelium was a key site of influence for L-NNA (intact) and Gap 27 (Ovx). Thus present and previous results imply that the ADP response in pial arterioles represents the additive actions of an endothelial and a GL component. That supposition was confirmed in the present study by the finding that combining endothelial and GL injury produced an essentially complete loss of ADP reactivity in both intact and Ovx females. Finally, topical application of the selective P(2)Y(1) antagonist, MRS-2179, was associated with a nearly complete suppression of the ADP response in both intact and Ovx females. These results suggest that 1) ADP-induced pial arteriolar dilation involves additive contributions from P(2)Y(1) receptors present in both vascular endothelium and the GL; 2) the influence of the GL component is not altered by ovariectomy; and 3) the gap junction-dependent component of the ADP response in Ovx females is unlikely to include the GL and probably resides in the vessels themselves.