Beta-arrestins are a family of regulatory and scaffold proteins functioning in signal transduction of G protein-coupled receptors including opioid receptors. Upon agonist stimulation, beta-arrestins bind to opioid receptors phosphorylated by G protein-coupled receptor kinases and promote receptor internalization and desensitization. Studies indicated that beta-arrestins are required in the development of morphine tolerance in mice. In the current study, we investigated the potential regulatory effects of morphine administration on beta-arrestin 1 and beta-arrestin 2 mRNA levels in different brain regions in rat using in situ hybridization method. Our results showed that the acute morphine administration (10 mg/kg) resulted in approximately 30% reduction in both beta-arrestin 1 and beta-arrestin 2 mRNA levels in hippocampus while the chronic morphine treatment (10 mg/kg, b.i.d., for 9 days) caused no significant change in level of either beta-arrestin mRNA. In locus coeruleus, both acute and chronic morphine treatments resulted in significant decreases (over 50%) in beta-arrestin 1 mRNA level but failed to induce any change in the level of beta-arrestin 2 gene expression. The acute morphine administration had no significant effect on beta-arrestin 1 or beta-arrestin 2 mRNA level in periaqueductal gray and cerebral cortex. However, after chronic morphine treatment, beta-arrestin 2 mRNA level decreased by 40% in periaqueductal gray and increased by 25% in cerebral cortex, in strong contrast to the unchanged beta-arrestin 1 mRNA level in these two brain regions. Furthermore, spontaneous or naloxone-precipitated withdrawal of morphine that did not affect the level of beta-arrestin 1 mRNA resulted in an aberrant increase (100% over control) in beta-arrestin 2 mRNA level in hippocampus. Our results thus demonstrated for the first time that opiate administration regulates level of beta-arrestin mRNAs in brain and the expression of beta-arrestin 1 and beta-arrestin 2 subtypes is differentially regulated in locus coeruleus, periaqueductal gray, and cerebral cortex by morphine. These data suggest that beta-arrestin 1 and beta-arrestin 2 may play different roles in the development of opioid tolerance and dependence.