Structural studies of modified nucleobases in RNA duplexes are critical for developing a full understanding of the stability and specificity of RNA base pairing. 2-Thio-uridine (s(2)U) is a modified nucleobase found in certain tRNAs. Thermodynamic studies have evaluated the effects of s(2)U on base pairing in RNA, where it has been shown to stabilize U:A pairs and destabilize U:G wobble pairs. Surprisingly, no high-resolution crystal structures of s(2)U-containing RNA duplexes have yet been reported. We present here two high-resolution crystal structures of heptamer RNA duplexes (5'-uagcs(2)Ucc-3' paired with 3'-aucgAgg-5' and with 3'-aucgUgg-5') containing s(2)U:A and s(2)U:U pairs, respectively. For comparison, we also present the structures of their native counterparts solved under identical conditions. We found that replacing O2 with S2 stabilizes the U:A base pair without any detectable structural perturbation. In contrast, an s(2)U:U base pair is strongly stabilized in one specific U:U pairing conformation out of four observed for the native U:U base pair. This s(2)U:U stabilization appears to be due at least in part to an unexpected sulfur-mediated hydrogen bond. This work provides additional insights into the effects of 2-thio-uridine on RNA base pairing.