Nidoviruses produce an extensive 3'-coterminal nested set of subgenomic mRNAs, which are used to express their structural proteins. In addition, arterivirus and coronavirus mRNAs contain a common 5' leader sequence, derived from the genomic 5' end. The joining of this leader sequence to different segments (mRNA bodies) from the genomic 3'-proximal region presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis. Key elements in this process are the so-called transcription-regulating sequences (TRSs), which determine a base-pairing interaction between sense and antisense viral RNA that is essential for leader-to-body joining. To identify RNA structures in the 5'-proximal region of the equine arteritis virus genome that may be involved in subgenomic mRNA synthesis, a detailed secondary RNA structure model was established using bioinformatics, phylogenetic analysis, and RNA structure probing. According to this structure model, the leader TRS is located in the loop of a prominent hairpin (leader TRS hairpin; LTH). The importance of the LTH was supported by the results of a mutagenesis study using an EAV molecular clone. Besides evidence for a direct role of the LTH in subgenomic RNA synthesis, indications for a role of the LTH region in genome replication and/or translation were obtained. Similar LTH structures could be predicted for the 5'-proximal region of all arterivirus genomes and, interestingly, also for most coronaviruses. Thus, we postulate that the LTH is a key structural element in the discontinuous subgenomic RNA synthesis and is likely critical for leader TRS function.