Deletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1), strain 17 syn+, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and Bg/II restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with Bg/II and nuclease BAL 31 followed by ligation and recleavage with Bg/II resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following Bg/II and SstI treatment of pTK1 were recombined with wild-type (wt) HSV-1 (17) syn+ DNA in baby hamster kidney (BHK) cells to produce TK- deletion mutants HSV-1 (17) TK 1301, HSV-1 (17) TK 1302 and HSV-1 (17) TK 1303 respectively. 5-Bromo-2'-deoxyuridine, 5-bromo-2'-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK+ virus in heterogeneous recombinant stocks analysed for the presence of TK- recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-1 (17) TK 1302 were TK-, failed to produce polypeptides of molecular weights 43000 and 19000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK- mutant HSV-1 (17) dPyk-7. Superinfection studies with HSV-1 (17) TK 1301, HSV-1 (17) TK 1302, HSV-1 (MDK) and HSV-1 (17) dPyk-7 indicated that all TK- mutants except dPyK-7 produce a trans-acting gene product which can switch on the transforming HSV-1 TK gene.