A series of cell lines was constructed by transformation of murine LTK- cells with a family of deletion mutants of the herpes simplex virus (HSV) thymidine kinase (tk) gene. These mutants, differing in the extent of 5' sequence flanking the coding region for tk, varied in the frequency with which they were able to convert tk- cells to the tk+ phenotype. Converted cell lines were analysed for tk DNA sequences, tk mRNA sequences, the 5' terminus of tk-specific transcripts and for their ability to respond to a signal provided in trans by infecting tk- virus (transactivation). The results of these analyses reveal that transformation efficiency correlates inversely with the extent of 5' flanking information. Thus mutants retaining less than 109 bp of 5' sequences transform less efficiently than those that retain at least 109 bp. Cell lines established by transformation with mutants retaining the proximal 109 bp contain relatively few copies of tk DNA whereas those which arose as a result of transformation with mutant DNA containing less than 109 bp generally contained multiple copies of tk DNA. Analyses of tk-specific transcripts revealed that cell lines derived from plasmids that transformed efficiently synthesized an mRNA which was indistinguishable by its size or 5' end from infected cell mRNA. Cell lines established by plasmids that were inefficient at transformation accumulated truncated mRNAs that initiated at aberrant start sites. The presence of the 5' 109 bp block was required for transformants to increase the level of tk mRNA and enzyme when infected with a tk- deletion mutant of HSV. We also show that transactivation does not alter the initiation site of the tk mRNA synthesized by transformants.