We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.