Background: Specific immunotherapy for respiratory allergy, a seasonal disease, significantly reduces the inflammatory process, attenuating the clinical symptoms. The mechanism for the clinical beneficial effect of immunotherapy has not yet been clarified. Melatonin shows a circadian and seasonal variation and together with the endogenous opioid system plays an immunomodulatory role acting on both specific and nonspecific immunity responses. Thus, the possibility that immunotherapy involved changes in the melatonin-opioid system was investigated. Methods: Thirty-five children aged 3–15 years with rhinitis and asthma due to olive + grass pollen sensitization were studied. The patients were treated with depot extracts containing the identified allergens with increasing doses from 1 to 1,000 IU/ml during 3 months. Melatonin, β-endorphin, total and specific IgE and IgG4 were determined before and after treatment. Results: All children showed a significant improvement of their symptoms at the end of the treatment, coinciding with a significant drop of both melatonin and β-endorphin levels. Total IgE decreased in most of the cases although the mean values did not show significant changes. Specific IgE and IgG4 were also unchanged. A significant correlation between melatonin and β-endorphin and between β-endorphin and IgG4 was found before immunotherapy, and these correlations disappeared when the treatment was finished. Conclusions: The decrease in the levels of melatonin and β-endorphin suggests the disappearance of their stimulating influence on the immune system. Thus, hyposensitization after immunotherapy may involve, at least in part, the inhibition of the immunoenhancing properties of the melatonin-opioid system.