Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro and in vivo, the occurrence of transduced cells resistant to GCV due to a deletion within HSV-tk. This deletion, a consequence of the presence of cryptic splice donor and acceptor sites, originates in the retroviral producer cell. Here we adopt two different methods that introduce third-base degenerate changes at the cryptic splice sites and so prevent splicing. Consequently, the HSV-tk protein is unaltered and the sensitivity of the target cells to GCV is preserved. The use of this mutated HSV-tk should reduce the likelihood of the development of resistant genetically modified cells during clinical trials.