Five thymidine kinase (TK)-deficient mutants of varicella-zoster virus derived from the Oka vaccine strain were examined to determine the state of transcription of the TK gene, and the DNA sequences of two of the mutants and the parent strain were investigated. Restriction endonuclease analysis revealed no major alteration of whole genome organization, but did reveal a minor change of the cleaved fragment in size. Northern blot analysis showed that the transcripts from the TK gene of all five mutants were of the same size as that of the parent strain (1.6 kb). DNA sequences of the TK gene of the parent strain and those of two mutants were determined. One had a base substitution (C to T) in the coding region of TK, resulting in the termination of amino acid elongation in the 68th amino acid of 341. The other had a two-base deletion (AT) in the coding region, resulting in a frame shift from the 126th amino acid and termination at the 162nd amino acid. Both mutants had premature stop codons which might result in the synthesis of nonfunctional TK polypeptides. Thus, the molecular basis of TK deficiency was determined.