The complete nucleotide sequence of the ns2 gene of human coronavirus OC43 (HCV-OC43) was determined. Sequence analysis revealed an open reading frame that could encode a protein of 278 amino acids, with an estimated molecular mass of 32.2 kDa. Six potential phosphorylation sites are present but no sites of N-glycosylation were found. The amino acid sequence of the HCV-OC43 ns2 protein shows 92% identity with that of the Mebus strain of bovine coronavirus (BCV). However, a stretch of nine consecutive amino acids near the C terminus is completely different, causing it to be very hydrophilic, which contrasts with the hydrophobic nature of this region in BCV. As shown by immunofluorescence with a monospecific antiserum, the ns2 protein was expressed in the cytoplasm of HCV-OC43-infected HRT-18 cells.