Psoralen is an asymmetric photoreactive intercalator with a furane and a pyrone side. When intercalated at 5'-TpA-3' sites and upon UVA irradiation, the psoralen can react with the thymine residues on both strands, introducing an interstrand crosslink. Using psoralen-coupled triple-helix-forming oligonucleotides, psoralen interstrand crosslinks can be site-specifically introduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors. In addition, crosslinks introduced via a triple-helix-forming oligonuleotide are oriented with the furane side of the psoralen associated with a specific strand of the target sequence. Here, the transformation efficiency, the mutation frequency and the mutational spectra of site-specifically placed and oriented crosslinks were examined in yeast cells. We found that the nature of the targeted mutations depended on the crosslink orientation: bypass of the pyrone-adducted thymine yielded T-->A or T-->C substitutions and A insertions, while bypass of the furane-adducted thymine yielded T-->G substitutions and G insertions. Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis. In addition, the observed pattern of mutagenesis suggests a coupling to transcription, similar to the one observed in mammalian cells. Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed strand, suggesting that the incision preference of psoralen crosslinks, which has been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells.