A total of 102 blood samples were used in a prospective parallel and blind study to evaluate three commercially available anti-pp65 monoclonal antibodies for cytomegalovirus antigenemia assay, at the dilutions recommended by their manufacturers. Cytomegalovirus was detected in 42 samples (41.2%), by either culture (32 samples; 76.2% of positive samples) or antigenemia (38 samples; 90.6%). Of the antigenemia-positive samples, 37 were detected by Monofluo kit CMV, which showed statistically significant differences when compared with the other reagents (Biosoft 1C3 and Clonab C10/C11), in either positivity rates (P < 0.004) or positive cell counts (P < 0.001). This reagent also gave better results in fluorescence quality than 1C3 and C10/C11. However, technical differences were not reflected in the clinical relevance of the antigenemia results.