Two new immunological methods, the luminescent immunofocus assay (LIFA) and the luminescent immunofocus inhibition assay (LIF-IA), are described for the quantitation of cytopathic and non-cytopathic viruses propagated on cell culture monolayers. These methods use enhanced chemiluminescent detection to identify foci (luminescent immunofoci, LIF) of virus-infected cells. Viruses are propagated in susceptible cells under an agarose overlay, inactivated with ultraviolet irradiation, lifted onto nitrocellulose membranes, and probed with virus-specific monoclonal or polyclonal antibody followed by a second antibody conjugated to horseradish peroxidase. Membranes are then treated with a luminol-based detection reagent and exposed to light sensitive film for up to 10 min. The film is developed and foci appear as dark, discrete spots which are proportional to the dose of each virus. The LIFA detected both cytopathic and non-cytopathic hepatitis A viruses (HAV) and simian rotavirus. For the cytopathic HAV, the LIFA and plaque counts were comparable. The LIF-IA was developed for HAV using virus-specific antiserum which effectively attenuated LIF formation. The LIFA and LIF-IA may be completed 5 days faster than conventional radioimmunofocus assays for HAV and rotavirus and do not require the use of radiolabeled antibodies, offering safety advantages and making these techniques more adaptable for general use. Luminescent immunofocus assays should be useful for the detection and quantitation of virtually any cytopathic or non-cytopathic virus that can be propagated in monolayer cultures when virus-specific antiserum is available.