Previously, we have used the biochemical receptor binding method to investigate whether down-regulation of the opioid receptor is a mechanism for morphine tolerance, and we were led to a negative conclusion. In the current study, we further used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats (250-300 g) were chronically treated with morphine s.c. for 2, 4 or 6 days, using an escalating dosage paradigm (5-45 mg), which resulted in a 1.8 to 4.0-fold increase in AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu- or delta-opioid receptor immunohistochemistry using the Avidin-Biotin Complex (ABC) method. No significant decrease in mu-opioid receptor (MOR) immunodensity was found in most of the brain regions, which were enriched with MOR after chronic treatment with morphine except for the anteroventral thalamic nucleus in the ventrolateral part (AVVL). No significant change in delta-opioid receptor (DOR) immunodensity after chronic treatment with morphine was found either. Therefore, our conclusion is that down regulation of opioid receptors may not be an important mechanism for morphine tolerance.