In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).