SUMMARYWe have determined the herpes simplex virus (HSV) type 2 DNA sequences responsible for the initiation of morphological transformation and have investigated the retention and expression of these sequences in morphologically transformed cells and in tumours derived from these cells. All the transformed cells analysed were selected by a focus formation assay and are oncogenic in the inbred host rat. Cloned HindIII and BglII fragments from the HSV-2 genome were assayed for the ability to initiate morphological transformation of rat embryo cells. Only the HindIII a (map units 0.52 to 0-72) and the BgllI n (0'582 to 0.612) clones gave transformed foci. This shows that the BglII n region is responsible for initiation of transformation. Southern blot analysis of DNA extracted from these transformed cells and from turnouts derived from these transformed cells revealed that neither the BgllI n fragment nor fragments of 500 bp mapping within it are detected at the level of one copy per cell and therefore need not be retained in the cell to maintain the oncogenic phenotype. In addition there was no evidence of expression of the HSV-specified ribonucleotide reductase activity which is partially encoded within the BglII n fragment of HSV-2. We also analysed DNA from rat embryo cells transformed by ts mutants of HSV-2 (HG52) or HSV-1 (HFEM or 17) at non-permissive temperature or by virus at supraoptimal temperature or by sheared virus DNA and DNA from tumours derived from lines of these transformed cells. In addition, we cloned both transformed and tumour cell lines and analysed these similarly. In no case could we detect HSV DNA sequences at the level of one copy per cell.