We have previously shown that the nuclear membrane fluidity of rat liver, measured by fluorescence anisotropy of two probes, is higher in the hydrophobic core, with respect to the bilayer surface, in newborn rats compared to adult rats. The aim of the present research is to investigate whether the nuclear membrane fluidity influences RNA nucleocytoplasmic transport. To this end two experimental models were used: the fluidity of nuclear membrane isolated from adult rats was increased by a choline base exchange reaction, which is known to be accompanied by an increase of phosphatidylcholine unsaturated fatty acids, whereas that of nuclear membrane isolated from newborn rats was decreased by incubation with dimyristoylphosphatidylcholine-cholesterol liposomes. The RNA efflux, evaluated by using [3H]uridine, significantly increased in the adult nuclear membrane submitted to choline base exchange reaction, whereas a strong decrease in the newborn nuclear membrane enriched with cholesterol was found. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme which is correlated with mRNA transport, showed parallel variations. Therefore, for the first time, we have provided evidence that the nuclear membrane fluidity plays a regulatory role in RNA nucleocytoplasmic transport, although the mechanism by which this effect takes place remains to be clarified.