An antiserum against the crustacean pigment-dispersing hormone (PDH) was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila melanogaster and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla-the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons)-that seem to be involved in the control of adult circadian rhythmicity. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and of four neurons with small somata (small PDFMe neurons). Both subgroups were usually absent in adults of behaviorally arrhythmic mutants of disconnected. In the wild type, PDH immunoreactivity was seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons were found to persist unchanged into adulthood, whereas the large ones seemed to develop halfway through metamorphosis. Beside the PDFMe neurons, three other clusters of PDH-immunoreactive neurons were stained in the developing nervous systems of Drosophila and are described in detail. Two of them were located in the brain, and the third was located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons were absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults.