Chronic administration of methamphetamine (MAP) up-regulated the mRNA expression of diazepam binding inhibitor (DBI) in rat brain, possibly leading to anxiety. Acute effects of MAP on anxiety associated with DBI, however, are not clear. In this study, we examined the effects of acute administration of MAP on behavior related to anxiety and the expression level of DBI mRNA and pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA, calibrated with the glyceraldehydes 3-phosphate dehydrogenase mRNA as the internal control in rat brain. The elevated plus-maze test was applied to the analysis of the possible anxiety-related profile of MAP. Acute administration of MAP (5 mg/kg, intraperitoneal administration) significantly increased spent time in the open-space arms at 4 h after the administration compared with a saline-treated group. The expression of DBI mRNA in a large number of regions of rat brain significantly decreased 2, 4, 8 and 16 h after acute administration of MAP. In contrast, the expression of PACAP mRNA in a large number of regions of rat brain significantly increased 4 and 8 h after the administration of MAP. These results suggest that MAP, at this dose, has an anxiolytic effect, based on the reduction of the putative anxiogenic peptides, DBI.