The core nucleotide sequence of bovine (Bos taurus) testicular PH-20 hyaluronidase was cloned using one step RT-PCR. The 5' and 3' regions were cloned separately and a sequence overlap of 124 bp facilitated the fusion of these two fragments by overlapping PCR, resulting in a concatenated sequence of 1422 bp. This nucleotide sequence and its deduced amino acid sequence were compared to homologous sequences from eight other mammal species. The bovine sequences were most similar to those of the pig, Sus scrofa (swine Spam1: 79.1% nucleotide and 70.1% amino acid similarity) and least similar to sequences from the Norway rat, Rattus norvegicus (murine Spam1: 61% nucleotide and 53.3% amino acid similarity). A phylogenetic analysis joined the red fox (Vulpes vulpes) sequence as sister to the bull-pig pair. Twelve cysteine residues were conserved among all nine aligned amino acid sequences and five proposed glycosylation sites have been identified. The feasibility of developing an effective, low-cost bovine PH-20 expression system is discussed in light of these new data.