NcSUB1 (formerly known as NC-p65) is the first molecularly described proteolytic enzyme of the intracellular protozoan parasite Neospora caninum. This report describes the characterization of a rabbit anti-N54, which is an antiserum generated against an internal fragment of NcSUB1 (amino acids 649-783). In immunofluorescence studies rabbit and-N54 labeled the apical end of the fixed parasite. By immuno-gold electron microscopy, the antibody bound primarily to the microneme organelles of the parasite. Analysis of secreted parasitic proteins indicated that a protein of molecular weight 65 kDa (reduced) or 55 kDa (nonreduced) was recognized bythe antibody. The same secreted proteins were affinity purified with rabbit anti-N54-coupled resins and were shown to contain major proteolytic activity by zymography. Thus, rabbit anti-N54 is the first antibody developed for N. caninum that binds to themicroneme proteins and recognizes a major secreted enzyme.