Several sets of clock neurons cooperate to generate circadian activity rhythms in Drosophila melanogaster. To extend the knowledge on neurotransmitters in the clock circuitry, we analyzed the distribution of some biogenic amines in relation to identified clock neurons. This was accomplished by employing clock neuron-specific GAL4 lines driving green fluorescent protein (GFP) expression, combined with immunocytochemistry with antisera against serotonin, histamine, and tyrosine hydroxylase (for dopamine). In the larval and adult brain, serotonin-immunoreactive (-IR) neuron processes are in close proximity of both the dendrites and the dorsal terminals of the major clock neurons, the s-LN(v)s. Additionally, the terminals of the l-LN(v) clock neurons and serotonergic processes converge in the distal medulla. No histamine (HA)-IR processes contact the s-LN(v)s in the larval brain, but possibly impinge on the dorsal clock neurons, DN2. In the adult brain, HA-IR axons of the extraocular eyelet photoreceptors terminate on the dendritic branches of the LN(v)s. A few tyrosine hydroxylase (TH)-IR processes were seen close to the dorsal terminals of the s-LN(v)s, but not their dendrites, in the larval and adult brain. TH-IR processes also converge with the distal medulla branches of the l-LN(v)s in adults. None of the monoamines was detectable in the different clock neurons. By using an imaging system to monitor intracellular Ca(2+) levels in dissociated GFP-labeled larval s-LN(v)s, loaded with Fura-2, we demonstrated that application of serotonin induced dose-dependent decreases in Ca(2+). Thus, serotonergic neurons form functional inputs on the s-LN(v)s in the larval brain and possibly also in adults.