The cauliflower mosaic virus (CaMV) 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600-nt leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and packaging. We have recently documented that the 35 S RNA leader adopts an elongated hairpin conformation and additional higher-order structures, a long-range pseudoknot and a dimer. Alternative structures might coexist, probably fulfilling specialized functions. In this paper, we analyze the biological significance of the elongated hairpin structure. We have introduced a spectrum of large deletions and small substitutions in the 35 S RNA leader and characterized their impact on the structure by temperature gradient gel electrophoresis. This analysis showed that the elongated hairpin consists of three sections of different stability (stem section I, II, and III). The overall secondary structure is relatively stable in the range of 10–32°C. It melts between 32 and 38°C in a manner indicating that the most stable base pairing occurs at the base of the elongated hairpin (stem section I). Mutations that destabilize stem section I decrease both the melting temperature of the leader and the expression in vitro and in vivo of the downstream CAT-reporter gene. Compensatory mutations restoring the stable elongated hairpin upregulate the translation efficiency. Our results demonstrate that the regulation of translation of the CaMV 35 S RNA is mediated by a stable hairpin in the leader.