A novel "sandwich" immunoassay for hepatitis B surface antigen monitored by chemiluminescence is described. The method involves use of an antibody-coated microtitration plate and requires 100 micro L of test specimen. The antigen binds to the antibody during the first 2-h incubation and, after an intermediate wash step, the sandwich is completed by 2-h incubation with antibody to antigen that has been labeled with an isoluminol derivative. A final wash step follows. A luminometer, built in-house, adds "microperoxidase" and peroxide, to initiate chemiluminescence, and provides automated readout at 10-s intervals. Results compare well in specificity and sensitivity with those of a comparison radioimmunoassay procedure. Within- and between-assay variability (CV) is 7 to 13% (n = 6). All reagents are stable at 4 degrees C for at least several months. Use of a non-radioisotopic label in this assay avoids the stability problems and inconvenience associated with radioactivity.