The carbonic anhydrase inhibitor acetazolamide causes extracellular acidosis and dilatation of cerebral arterioles. In this study we tested the hypothesis that acetazolamide also may induce headache and dilatation of cranial arteries. In a randomized double-blind crossover study design, 12 young healthy women were allocated to injection of 1 g acetazolamide or placebo on two separate days. We recorded headache on a verbal rating scale from 0-10 during an immediate phase (0-90 min) and a delayed phase (2-12 h). The circumference of cranial arteries was measured using 3T high-resolution magnetic resonance angiography 30 and 60 min after injection. Acetazolamide provoked immediate headache in 9 participants compared to 3 participants after placebo (P = 0.031). 11 participants reported headache in the delayed phase after acetazolamide compared with four after placebo (P = 0.016). The area under the headache curve was increased after acetazolamide compared to placebo in the delayed phase (2-12 h) (P = 0.005). Compared to placebo, arterial circumference increased after acetazolamide in the basilar artery (P = 0.002) aswell as the cerebral (P = 0.003), cavernous (P = 0.002) and cervical (P = 0.005) parts of the internal carotid artery, but no other extracranial arteries changed after acetazolamide. In conclusion, acetazolamide caused immediate and delayed headache as well as dilatation of intracranial arteries in healthy volunteers. It is possible that extracellular acidosis induced by acetazolamide causes sensitization of cephalic perivascular nociceptors, which, in combination with vasodilatation, leads to delayed headache.