RNA interference (RNAi) is an evolutionarily conserved eukaryotic adaptive response that leads to the specific degradation of target mRNA species in response to cellular exposure to homologous double-stranded RNA molecules. Here, we have analyzed the subcellular location at which RNA degradation occurs in human cells exposed to double-stranded short interfering RNAs. To unequivocally determine whether a given mRNA is subject to degradation in the cytoplasm, the nucleus, or both, we have used the retroviral Rev/RRE system to control whether target mRNAs remain sequestered in the nucleus or are exported to the cytoplasm. In the absence of export, we found that the nuclear level of the RRE-containing target mRNA was not affected by activation of RNAi. In contrast, when nuclear export was induced by expression of Rev, cytoplasmic target mRNAs were effectively and specifically degraded by RNAi. Curiously, when the target mRNA molecule was undergoing active export from the nucleus, induction of RNAi also resulted in a reproducible approximately twofold drop in the level of target mRNA present in the nuclear RNA fraction. As this same mRNA was entirely resistant to RNAi when sequestered in the nucleus, this result suggests that RNAi is able to induce degradation of target mRNAs not only in the cytoplasm but also during the process of nuclear mRNA export. Truly nucleoplasmic mRNAs or pre-mRNAs are, in contrast, resistant to RNAi.