The distribution of a CRF-related diuretic peptide (Musca-DP) and the diuretic/myotropic insect myokinins in the central nervous system of larval and adult houseflies was investigated using antisera raised against Locusta-DH and leucokinin-I, respectively. Two separate, small populations of immunoreactive neurons are present in the brain and fused thoracic-abdominal ganglion mass. There is no evidence for these immunoreactivities being colocalised either within single neurons or at neurohaemal release sites. Crude extracts of tissues containing immunoreactive material increase fluid secretion by isolated Malpighian tubules from adult flies. Diuretic activity is highest in tissues containing myokinin-immunoreactive material. Consistent with this observation, myokinin analogues produce a four- to five-fold increase in fluid secretion, which is more than twice the response to Musca-DP. These effects are mimicked by treatments that increase intracellular calcium and cyclic AMP, respectively. When tested at threshold concentrations, the two classes of diuretic peptide act synergistically to accelerate tubule secretion, and their separate localisation may be important for the precise control of diuresis.