Melatonin, an indoleamine hormone synthesized in the pinealocytes, is electroactive at the surface of pre-treated carbon fibre microelectrodes (mCFE)in vitro when using differential-pulse voltammetry (DPV), at the specific oxidation potential of approximately +570 mV. In vivo DPV experiments have then been performed in melatonergic regions such as the pineal gland or the suprachiasmatic nucleus (SCH) of anaesthetized adult male rats. These experiments indicated the feasibility of simultaneous measurements of the indolaminergic peak 3, which occurred at approximately +280 mV, due mainly to the oxidation of extracellular 5-hydroxyindoleacetic acid (5HIAA), and a signal at approximately +580 mV which we called peak M. Pharmacological in vivo experiments performed in anaesthetized rats prepared for DPV analysis with the mCFE implanted into the pineal gland or the SCH indicated that intravenous or intra-cerebral injections of exogenous melatonin (5 mg kg^–1 or 2 µg µl^–1, n= 3, respectively) were followed by a selective and significant increase of in vivo peak M. Other in vivo experiments with anaesthetized rats prepared for DPV analysis with the mCFE into the SCH showed that tryptophan [TRY, 30 mg kg^–1 intravenous (i.v.), n= 3] and n-acetyl serotonin (nA-5HT, 5 mg kg^–1 i.v., n= 3), both precursors of melatonin, were responsible for a transient but significant increase in the size of peak M (approximately 320% or 126% of control levels within 10 min or 20 min, respectively). The second treatment was also responsible for an increase in the size of peak 3 (216% of control values within 15 min) which may have come from an alternative route of degradation of nA-5HT into the SCH, as supported by the increased levels of both peak 3 and peak M following local injection of exogenous 5HT (1 µmol dm^–3, n= 3) into the SCH. N-acetyltransferase (NAT) is one of the key enzymes controlling the synthesis of melatonin and its activity is stimulated by butyrate. Treatment of anaesthetized rats with sodium butyrate (100 mg kg^–1 i.v., n= 3) produced a transient increase in the size of in vivo peak M and peak 3. In conclusion, these data suggest that DPV with pre-treated microbiosensors might be the first in vivo voltammetric method for analysis of melatonergic function(s) in melatonergic brain areas.