The temperature dependence of ATP-enhanced RNA delivery from rat liver nuclei to a surrogate cytoplasm was investigated. Examination of linear-rate data on Arrhenius graphs of 1/T vs. log (% RNA delivered per min) revealed an activation energy of 12.5--13 kcal/mol. When data derived from longer incubation periods was displayed on Arrhenius graphs, we observed a discontinuous graph--two distinct linear segments with slopes of differing sign which intersected near 20 degrees C. It was demonstrated that this discontinuity was not due to lipid phase transition in the nuclear membranes and that its position depended upon treatment of the nuclei and upon additives to the incubation mixtures. The decline in transport apparent in the upper-temperature domain on 20-min Arrhenius graphs was shown to be based on the diffusion of transported macromolecular RNA back into the nucleus--a process greatly amplified by the rapidity of transport in this domain. The large net inward diffusion, in concert with significantly differing activation energies for RNA transport and passive diffusion, suggests that the process of nucleocytoplasmic RNA transport is not diffusion driven. Our data have established that an integral parameter of RNA transport (namely, the activation energy) remains unchanged in various in vitro manipulations.