The effects of corticotropin-releasing factor (CRF)-related (ZooneDH),calcitonin (CT)-related (RhoprDH31) and kinin-related (leucokinin I) peptides on the ion composition of fluid secreted by upper Rhodnius prolixus Malpighian tubules and on KCl reabsorption by the lower tubules were assessed. ZooneDH stimulated fluid secretion while increasing the[Na+] of secreted fluid at the expense of [K+]. Upper tubules responded to ZooneDH with a characteristic triphasic change in the transepithelial potential (TEP), reminiscent of the response to 5-hydroxytryptamine (5HT). RhoprDH31 produced a small (∼9 mV)lumen-positive shift in TEP of the upper tubule but had no effect on the rate of fluid secretion or ion composition of the secreted fluid. In contrast to 5HT, both peptides failed to activate KCl reabsorption by the lower tubule. Leucokinin I had no effect on the ion composition of fluid secreted by whole or upper Malpighian tubules. We propose that: (1) 5HT and a native CRF-related peptide similar to ZooneDH activate the same second messenger systems and ion transporters in the upper tubule cells; (2) CRF-related peptide is utilized to maintain high rates of fluid secretion during the post-feeding diuresis and is additionally used at times when KCl reabsorption is unnecessary or detrimental. The differential actions of multiple diuretic factors allows for intricate control of ionic and osmotic balance in R. prolixus.