Melatonin production in extra-pineal sites is modulated by pathogen-associated molecular patterns and affects immune response. We investigated melatonin production by pineal vs. extra-pineal tissues of toads (Rhinella icterica). The animals were intraperitoneally injected with LPS (2mg/kg) or saline at 10h and 22h. Tissues from these toads (blood, bone marrow, lungs, liver, and intestine) were sampled 2 hours after the injection (noon and midnight). Plasma and tissue homogenates melatonin levels were determined by ELISA kits. LPS treatment increased melatonin concentration in bone marrow during the day, and melatonin levels in the bone marrow were higher during the day than at night in the LPS group. Melatonin concentration in the liver showed a tendency for higher concentration in the LPS than in the saline-injected toads during the night. Melatonin levels in the intestine were also higher at night than during the day. Plasma melatonin levels and lung melatonin concentration were not affected either by the treatment or the period. Our results showed that melatonin is present in extra-pineal tissues in R. icterica toads, and its concentration is modulated in different tissues by an immune challenge. Moreover, the period of increased melatonin production in response to LPS differs between tissues, evidencing the complexity of individual defense against pathogens.