This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH31, has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH37 in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH46 and Dippu-DH46 fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH37 increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.