The ability to simultaneously isolate intact DNA-free RNA, genomic DNA, and proteins from a biological specimen can be useful in cloning genes and analyzing gene expression. Equilibrium density gradient centrifugation with CsCl is a useful tool for fractionating, quantitatively separating, and characterizing RNA, DNA, and the total quota of proteins, respectively, based on differences in their buoyant densities. In the present study we have reexamined the rarely used cesium salt, cesium trifluoroacetate, for the same purpose. A significant advantage of CsTFA lies in the fact that, unlike in CsCl, RNA can be recovered from a single, soluble fraction of the CsTFA gradient. Furthermore, unlike CsCl, CsTFA is freely soluble in ethanol so that co-precipitation of the salt in the recovered RNA upon alcohol precipitation does not take place. Hence, the RNA is recovered with minimum manipulations. The one-step separation of cellular macromolecule classes free of each other in small amount of starting materials provides a major advantage over other methods currently in use. (C) 2003 Elsevier Inc. All rights reserved.