We examined the binding characteristics of unoprostone isopropyl and its metabolite, M1 (M1), in bovine corpus luteum membranes, mobilization of intracellular calcium in human ciliary muscle cells and cyclic AMP generation in rabbit iris-ciliary body. The ligand binding assay of 3H-unoprostone isopropyl and M1 did not demonstrate any specific binding of these compounds in the bovine corpus luteum membranes. However, there was a high specific binding of prostaglandin F2alpha. Competitive ligand binding studies showed that neither the docosanoid, unoprostone isopropyl, nor M1 binds to prostaglandin receptor sites. In human ciliary muscle cells that express EP1, EP2 and FP receptors, unoprostone isopropyl did not increase the mobilization of intracellular calcium nor was it able to generate cyclic AMP at low concentrations in rabbit iris-ciliary body. Similar observations were made with M1 on the above signal transduction pathways. From these results, it is concluded that unoprostone isopropyl and M1 do not bind to prostaglandin (PG) receptor sites in the bovine corpus luteum membranes and do not have affinity for PG receptors linked to intracellular calcium and cyclic AMP second messenger systems.