A new method for measurement of transbilayer distribution of sterol in plasma membranes is reported. The procedure utilized a fluorescent sterol, dehydroergosterol, and a chemical quenching agent, trinitrobenzenesulfonic acid. Dehydroergosterol was useful as a probe molecule for sterols for the following reasons, (a) Dehydroergosterol contained no bulky side chains as reporter groups. (b) Dehydroergosterol structurally resembled cholesterol and desmosterol, the primary sterol synthesized by LM fibroblasts. (c) Dehydroergosterol interacted with digitonin, filipin, and served as a substrate for cholesterol oxidase. (d) The phase transition of dipalmitoylglycerophosphocholine was completely abolished by dehydroergosterol. (e) The native sterol of LM fibroblasts, desmosterol, was completely replaced by dehydroergosterol without effect on LM cell growth, cell doubling time, plasma membrane (Na+, K+)-ATPase and 5'-nucleotidase activity, microsomal NADPH-dependent cytochrome c reductase activity, and mitochondrial succinate-dependent cytochrome c reductase activity. (f) Neither the phospholipid composition nor the sterol/phospholipid ratio of LM fibroblasts were altered by supplementation with dehydroergosterol. The trinitrophenyl group of trinitrophenylglycine or of surface membranes of LM fibroblasts or red blood cells treated with trinitrobenzenesulfonic acid was an excellent quencher of dehydroergosterol fluorescence. Fluorescence in mouse very-low-density lipoproteins, LM fibroblasts plasma membranes, red blood cell surface membranes, and in rat red blood cell membranes was quenched 95 +/- 3%, 20 +/- 2%, 75 +/- 4%, and 69 +/- 4% respectively when the quenching agent was present on only the extracellular site of the membrane. Trinitrophenyl residues effectively quenched the dehydroergosterol fluorescence in the plasma membrane of LM cells by 20% when dehydroergosterol was present from 1-85 mol/100 ml of the membrane sterol. When both sides of the plasma membrane were trinitrophenylated, greater than 95% of the dehydroergosterol fluorescence was quenched. In addition, when LM cells were cultured with dehydroergosterol, exposed latex beads, and the endocytosed particles isolated as phagosomes and treated with trinitrobenzenesulfonic acid under non-penetrating conditions, the fluorescence of the dehydroergosterol was quenched nearly 64%. From these and other results we deduced that the inner monlayer of the LM fibroblasts plasma membrane was enriched with dehydroergosterol. In contrast, the distribution of the sterol in red blood cell membranes indicated an enrichment in the outer monolayer.