Abstract The NC-p65 cDNA is the first protease sequence cloned and described for Neospora caninum. The full length cDNA was isolated by 5′- and 3′-rapid amplification of cDNA ends (RACE). NC-p65 was composed of 865 amino acids with a predicted signal sequence, a proposed pro-domain, and an internal region of conserved repeats. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) and had a predicted active site made up of the catalytic residues, Asp 253, His 309, and Ser 484. Antibodies to recombinant NC-p65 recognized multiple bands on Neospora lysate immunoblots, but most intensely stained a 65 kDa band. When N. caninum proteins were purified with affinity resins specific for NC-p65 and analyzed for enzyme activity, a single specific band of reaction was observed on gelatin-saturated zymograms.