Flavivirus particles are synthesized in an immature form containing heterodimers of the proteins prM and E. Shortly before release from the cell, prM is cleaved by the host protease furin to yield mature virions. In this study, the furin-mediated cleavage of the tick-borne encephalitis (TBE) virus protein prM was prevented by specific mutagenesis of the cleavage site. This resulted in the production of immature TBE virions, which were shown to be completely non-infectious in BHK-21 cells. This finding contrasted with previous studies in which immature flavivirus particles produced by other techniques were shown to have considerable residual infectivity. The structural integrity of the mutant virus particles was confirmed by the characterization of physical and antigenic properties. Most importantly, infectivity could be restored by the addition of trypsin, which presumably cleaved protein prM at one of the monobasic sites retained in the mutated sequence. In the presence of trypsin, the mutant could be passaged repeatedly in BHK-21 cells, but if the protease was removed, the activated particles could initiate only a single round of infection, which again generated non-infectious virus progeny. These observations provide evidence that the infectivity of flaviviruses depends on the endoproteolytic cleavage of protein prM, which probably has a regulatory function rather than a direct role in virus entry. Moreover, the results illustrate that mutation of the furin cleavage site is a convenient way to produce single-round infectious flavivirus particles, which may be useful in vaccine and vector development.