Rubella virus genomic RNA contains a 5' stem-loop (5'(+) SL) which is required for efficient translation and replication. The La autoantigen previously was shown to bind this RNA sequence in vitro. Results reported here demonstrate that this cellular RNA-binding protein binds to the RV 5' SL RNA with sufficient specificity for the binding to occur in the presence of excess total cellular RNA. Further, the affinity of purified La for the RV sequence is similar to its affinity for known cellular substrates. To address the functional significance of La binding, mutant forms of the RV 5'(+) SL were analysed which bind La weaker or stronger than the native form. These three forms of the RV 5' SL were incorporated into RV-luciferase constructs which expressed luciferase activity in transient transfection. The level of expression from each construct correlated with the ability of its RV sequence to bind La. The detection of La/RV RNA complexes in infected cells further supported the physiological relevance of this interaction. Possible implications of autoantigen La interaction with RV RNA for rubella virus pathology and vaccine associated adverse reactions are discussed.