The use of monoclonal antibodies in the development of a third generation microtitration plate enzyme immunoassay for the detection of hepatitis B virus (HBV) is described. These antibodies were tested either alone or in combination with polyclonal antibodies raised in rabbit. Horseradish peroxidase was employed in the conjugates and 3,3',5,5'-tetramethylbenzidine was used as the enzyme substrate. Effect of type and concentration of antibodies used in the coating solution and in preparing conjugates as well as reaction time are discussed. The designed test employs a 100-microliters sample, with an overall incubation time of 3.5 hr. Serum or plasma (EDTA or citrate as anticoagulant) can be used. The sensitivity limit of the test was 0.4 ng/ml for subtype ad and 0.5 ng/ml for subtype ay. When used as screening test, 99.6% specificity was obtained; predictive value was 97.5% for a positive result and 99.8% for a negative result. According to the performance of the test, it seems to be suitable for diagnosis in routine laboratory and screening in blood banks.