Low and high frequency electroacupuncture (EA)-produced analgesia have been shown to be mediated by different brain substrates and different opioid peptides. In this study, Fos-like immunoreactivity (FLI) and in situ hybridization of the three opioid mRNAs were used to examine the effect of low (2 Hz) and high (100 Hz) frequency EA on neuronal activities, and the expression of opioid genes. 2 Hz and 100 Hz EA induced a markedly different spatial patterns of Fos expression in the rat brain, suggesting there are distinct neuronal pathways underlying EA of different frequencies. Likewise, 2 Hz and 100 Hz EA exert differential effects on opioid gene expression: while 2 Hz EA induced a more extensive and intensive preproenkephalin (PPE) mRNA expression than 100 Hz EA, it had no effect on preprodynorphin (PPD) mRNA expression which was significantly increased by 100 Hz EA stimulation. In contrast, EA of both frequencies did not affect POMC mRNA expression.