1 Prostacyclin and its stable analogue, carbacyclin, bind competitively to a single population of receptors, and activate adenylate cyclase of the NCB-20 neuronal somatic cell hybrid (Kact = 40.1 nM and 96.1 nM respectively). 2 Culture of NCB-20 cells in the presence of 1 microM carbacyclin for 4 to 16 h results in a progressive decrease in the prostacyclin-dependent activation of adenylate cyclase in cell homogenates with an increase at 16 h of the Kact from 64.1 nM to 174.0 nM and decrease in the maximum adenylate cyclase activation from 41.2 to 15.1 pmol cyclic AMP min-1 mg-1 protein. 3 The prediction that the apparent decrease in affinity in the prostacyclin-dependent activation of adenylate cyclase was secondary to a reduction in receptor numbers was tested directly by measuring binding of [3H]-prostacyclin to membranes of cells exposed to carbacyclin for 16 h. This showed an actual decrease in affinity of the prostacyclin-receptor interaction, as well as a decrease in the total receptor numbers. Thus prolonged exposure of NCB-20 cells to carbacyclin caused reductions in both receptor numbers and affinity, reflected by measurements both of binding and adenylate cyclase activation.