Prior to the release of flavivirus particles from infected cells, the viral surface protein prM is cleaved to M by the cellular enzyme furin. For dengue virus (DENV), this maturation process appears to be very inefficient since a high proportion of progeny virions contain uncleaved prM. Furthermore, it has been reported that prM-containing DENV particles are infectious. These observations contradict the general assumption that prM processing is required to render virus particles infectious. Therefore, in this study, we reinvestigated the infectious properties of immature DENV virions. DENV particles were produced in furin-deficient LoVo cells. We observed that DENV-infected LoVo cells secrete high numbers of prM-containing particles. Subsequent analysis of the infectious titre revealed that immature particles lack the ability to infect cells, the infectious unit to particle ratio being 10 000-fold reduced compared with that of wild-type virus. Our results indicate that cleavage of prM to M is required for DENV infectivity.