NG108-15 mouse neuroblastoma x rat glioma cells were treated with the prostanoid IP receptor agonist iloprost (1 microM) and the time course of changes in the levels of prostanoid IP receptors, adenylyl cyclase activity, and the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein, Gs, were measured. Incubation of cells with iloprost produced a biphasic time course of desensitisation of prostanoid IP receptor-activated adenylyl cyclase. Parallel analysis of iloprost-induced loss of membrane Gs alpha, NaF-stimulated adenylyl cyclase and [3H]iloprost binding suggested only monophasic curves, with t0.5 values similar to the initial phase of desensitisation of iloprost-stimulated adenylyl cyclase activity. This suggests that the loss of receptor and Gs alpha occur at the same time and account for the initial period of desensitisation due to iloprost pretreatment. Pretreatment of NG108-15 cells with cholera toxin produced a near complete loss of membrane-associated Gs alpha, but the loss of [3H]iloprost binding due to iloprost treatment was not affected by pretreatment with cholera toxin, suggesting that prostanoid IP receptors can be down-regulated in the absence of any coupling to Gs. The second phase of desensitisation of iloprost-stimulated adenylyl cyclase activity, during which there was no further change in NaF-stimulated adenylyl cyclase or in the membrane levels of Gs alpha, was not due to protein kinase A activation, since elevating intracellular cyclic AMP levels with forskolin did not subsequently decrease iloprost-stimulated adenylyl cyclase activity or [3H]iloprost binding. These results demonstrate that iloprost pretreatment of NG108-15 cells induces two distinct phases of desensitisation; an initial desensitisation due to concurrent loss of prostanoid IP receptors and Gs alpha, and then a further desensitisation by an as yet uncharacterized mechanism during which there is no further loss of Gs alpha.