Many questions regarding the initiation of replication and translation of the segmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) remain to be solved. Computer analysis shows that the non-polyadenylated extreme 3'-untranslated regions (UTRs) of the coding strand of both genomic segments are able to fold into a single stem-loop structure. To assess the determinants for a functional 3'-UTR, we mutagenized the 3'-UTR stem-loop structure of the B-segment. Rescue of infectious virus from mutagenized cDNA plasmids was impaired in all cases. However, after one passage, the replication kinetics of these viruses were restored. Sequence analysis revealed that additional mutations had been acquired in most of the stem-loop structures, which compensated the introduced ones. A rescued virus with a modified stem-loop structure containing four nucleotide substitutions, but preserving its overall secondary structure, was phenotypically indistinguishable from wild-type virus, both in vitro (cell culture) and in vivo (chickens, natural host). Sequence analysis showed that the modified stem-loop structure of this virus was fully preserved after four serial passages. Apparently, it is the stem-loop structure and not the primary sequence that is the functional determinant in the 3'-UTRs of IBDV.