Urotensin II (UII) and its paralog urotensin II-related peptide (URP) are two vasoactive neuropeptides whose respective central actions are currently unknown. Here, we have compared the mechanism of action of URP and UII on cultured astrocytes. Competition experiments performed with [125I]UII showed the presence of very high- and high-affinity binding sites for UII, and a single high-affinity site for URP. Both UII and URP provoked a membrane depolarization accompanied by a decrease of input resistance, stimulated the release of endozepines, neuropeptides specifically produced by astroglial cells, and generated an increase in cytosolic calcium concentration ([Ca2+]c). The UII/URP-induced [Ca2+]c elevation was pertussis toxin (PTX)-insensitive, and was blocked by the PLC inhibitor U73122 or the IP3 channel blocker 2-APB. The addition of Ca2+ chelator EGTA reduced the peak and abolished the plateau phase whereas the T-type calcium channel blocker mibefradil totally inhibited the calcium response evoked by both peptides. However, URP and UII induced a mono- and biphasic dose-dependent increase in [Ca2+]c and provoked short- and long-lasting Ca2+ mobilization, respectively. Similar mono- and biphasic dose-dependent increase in [3H]inositol incorporation into polyphosphoinositides (PIP) in astrocytes was obtained but sole the effect of UII was significantly reduced by PTX, although BRET experiments revealed that both UII and URP recruited Go protein. Finally, UII exerted a dose-dependent mitogenic activity on astrocytes, but not URP. Therefore, we described that URP and UII exert not only similar but also divergent actions on astrocyte activity, UII exhibiting a broader range of activities at physiological peptide concentrations.