The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed inEscherichia colirevealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.