Antisera against the crustacean pigment-dispersing hormone (beta-PDH) were used in immunocytochemical preparations to investigate the anatomy of PDH-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and in that of several brain mutants of this species, some of which express altered circadian rhythmicity. In the wild-type and in all rhythmic mutants (small optic lobes, sine oculis, small optic lobes; sine oculis), eight cell bodies at the anterior base of the medulla (PDFMe neurons) exhibit intense PDH-like immunoreactivity. Four of the eight somata are large and four are smaller. The four large PDFMe neurons have wide tangential arborizations in the medulla and send axons via the posterior optic tract to the contralateral medulla. Fibers from the four small PDFMe neurons ramify in the median protocerebrum dorsal to the calyces of the mushroom bodies. Their terminals are adjacent to other PDH-immunoreactive somata (PDFCa neurons) which send axons via the median bundle into the tritocerebrum. The results suggest a possible involvement of the PDFMe neurons in the circadian pacemaking system of Drosophila. The location and size of the PDFMe neurons are identical with those of neurons containing the period protein which is essential for circadian rhythmicity. Changes in the arborizations of the PDFMe neurons in small optic lobes; sine oculis mutants are suited to explain the splitting in the locomotor rhythm of these flies. In the arrhythmic mutant, disconnected, the PDFMe neurons are absent. The arrhythmic mutant per0, however, shows normal PDH immunoreactivity and therefore, does not prevent the expression of PDH-like peptides in these neurons.