The P2Y6 receptor is a recently cloned P2 receptor which displays a high sensitivity for diphosphonucleotides. In 1321N1 astrocytoma cells stably expressing this receptor, UDP induced a slow and sustained accumulation of inositol trisphosphate via a pertussis toxin-insensitive G-protein: the maximal level was only reached after 15 min and a significant response was maintained for at least 3 h. A full second response to UDP was obtained after the first 45-min stimulation, but was lost after 165 min. This slow and sustained time-course and the lack of desensitization was reproduced with ADP. UTP was unable to restimulate the P2Y4 receptor, another recently cloned P2 receptor with a preference for UTP, after the first 5-min stimulation. The P2Y4 receptor is thus rapidly desensitized whereas desensitization of the P2Y6 receptor is delayed. The rank order of potency of various diphosphonucleotides at the P2Y6 receptor was: UDP > TDP > IDP > GDP > ADP >> CDP. The activity of three non-specific antagonists of P2 receptors was characterized by the following rank order of potency: reactive blue 2 > pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) > suramin. In conclusion, the most impressive features of the human P2Y6 receptor revealed by this study are the slow and sustained time-course of its activation and its high resistance to desensitization.