HCF-1 (host cell factor 1) is a human protein originally identified as a component of the VP16 transcription complex. A related protein HCF-2 is also present in humans and while at least HCF-1 appears to be required for normal cell growth there is currently little information on the precise cellular role(s) of these proteins. C. elegans contains a single HCF orthologue (CeHCF) which is very closely related to human HCF-2. To contribute to an understanding of the activities of these proteins here we analyse the subcellular localisation of the CeHCF protein in live transgenic worms and in mammalian cells. We constructed a green fluorescent protein (GFP) fusion of CeHCF and studied localisation after ectopic expression under the control of a heat shock protein promoter. The CeHCF-GFP protein accumulated in the cell nuclei at every stage of development and in a wide variety of cell types. Nuclear accumulation with nucleolar sparing was evident on the larvae and adult stages, but not earlier in development in which the protein accumulated diffusely in the nucleoplasm. Surprisingly the same protein accumulated in the mitochondria of a stable HeLa cell line, suggesting a differential localisation of CeHCF in mammalian cells. Furthermore, when overexpressed in transient transfection the CeHCF accumulated in both nuclear and mitochondrial compartments. We have refined the targeting determinants of CeHCF to the last 23 amino acids at the extreme C-terminus and show that they contain interdigitated amino acids involved in both nuclear and mitochondrial targeting. This novel targeting signal is sufficient to redirect HCF-2 into mitochondria. It can also be transferred to an unrelated protein, resulting in its targeting to both the mitochondrial and nuclear compartments.