The complete nucleotide sequence of the gene encoding the small subunit of ribonucleotide reductase (RNR) from bovine herpesvirus type-1 (BHV-1) was determined. The genomic DNA fragment sequenced also represented regions corresponding to the carboxy termini of RNR large subunit and of a virion protein causing host shut-off. The small subunit polypeptide was constituted of 314 amino acid residues totalling 35.25 kDa. The major transcription initiation and termination sites of the small subunit mRNA were located 95 bases upstream and 88 nucleotides downstream from the coding region, respectively. These findings indicate that the mRNA was 1128 bases long which correlated well with the size of the polyadenylated transcript detected in Northern blot analysis (1.3 kb). Within the RNR large subunit coding region, a TATA box and two CAAT box motifs were found 26, 104, and 190 nucleotides, respectively, upstream from the transcription initiation site of the small subunit mRNA. In contrast to previous studies (Slabaugh et al., J. Virol. 1988, 62, 519-527; Boursnell et al., Virology 1991, 184, 411-416), our comparative analysis of five herpesviruses, one iridovirus, and one poxvirus small subunit protein sequences suggested that the seven viruses arose from a common lineage.