Human rhinovirus (HRV) 3C protease (3Cpro) plays several important roles in the virus replication cycle. This enzyme cleaves the viral polyprotein at discrete sites to produce mature viral proteins and also inhibits cellular RNA transcription. It is not clear, however, whether the observed transcriptional shutoff activities are due to 3Cpro itself or to 3Cpro-containing precursors, and where 3Cpro exerts its effects within infected cells. To address these questions HeLa cells were infected with HRV-16, stained with polyclonal antibodies directed against 3Cpro and then analysed by laser confocal microscopy. Proteins containing 3Cpro accumulated in nuclei 2-4 h post-infection, and progressively increased in the cytoplasm. Analyses of subcellular extracts demonstrated that 3CD', a minor component among 3Cpro precursors, gave rise to the earliest 3Cpro nuclear signals. Mature 3Cpro and another 3Cpro precursor, 3CD, were also detected in the nucleus, cytoplasm and perinuclear membrane fractions 4 h post-infection. Transfecting cells with 3Cpro, 3CD precursor and 3CD(Delta371) (with deletion of 371 aa at the carboxyl terminus of 3D) demonstrated that the nucleolar localization signal was near the amino terminus of 3D. In addition, 3Cpro precursors were found to co-localize in nuclei with the transcription factor OCT-1 and the nucleolar chaperone B23. Finally, it was demonstrated that HRV-16 3Cpro, 3CD and 3CD(Delta371) could cleave OCT-1. Collectively, these findings suggest that HRV 3CD' and/or 3CD are specifically localized to the nucleoli of infected cells during the early stage of infection, and contribute to the inhibition of cellular RNA transcription via a proteolytic mechanism.