The identification of antivirals and vaccines against hepatitis C virus (HCV) infection is hampered by the lack of convenient animal models. The need to develop surrogate models has recently drawn attention to GB virus B (GBV-B), which produces hepatitis in small primates. In a previous study in vitro, it was shown that GBV-B NS3 protease shares substrate specificity with the HCV enzyme, known to be crucial for virus replication. In this report, GBV-B NS3 activity on GBV-B precursor proteins has been analysed in a cell-based system. It is shown that mature protein products are obtained that are compatible with the cleavage sites proposed on the basis of sequence homology with HCV and that GBV-B NS4A protein is required as a cofactor for optimal enzymatic activity. Experiments in vitro supported by a structural model mapped the region of NS4A that interacts with NS3 and showed that the GBV-B cofactor cannot be substituted for by its HCV analogue.