We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter. Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition. If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition. Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription. Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element. These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA.