1 [3H]-prostacyclin bound to a washed membrane preparation of the NCB-20 neuronal hybrid cell line. 2 Kinetic analysis of [3H]-prostacyclin binding suggested a simple, non-cooperative bimolecular interaction between the ligand and a single receptor population. The equilibrium dissociation constant was 16.6 nM, and binding at a saturating [3H]-prostacyclin concentration enabled the receptor density of 2.57 x 10(5) receptor molecules per cell to be calculated. 3 At 20 degrees C the rate constant for the forward reaction (K+1) was 2.26 x 10(5) M-1 S-1, and the rate constant for the dissociation of the ligand-receptor complex (k-1) was 3.85 x 10(-3) S-1. Thus the dissociation constant (k-1/k+1) was 17.0 nM. 4 Prostaglandin E1 and prostacyclin compete for a single receptor in these cells, and comparison of other prostaglandins as inhibitors of [3H]-prostacyclin binding revealed some of the structural requirements for high-affinity occupation of prostacyclin receptors.