The enzyme-linked immunosorbent assay (ELISA) enabled a rapid and accurate detection of potato virus Y (PVY), cucumber mosaic virus (CMV), alfalfa mosaic virus and tobacco mosaic virus in pepper. The sensitivity of this method made it possible to detect CMV or PVY: as early as 4 and 6 days after inoculation, respectively; directly, in leaf disks without prior homogenization; and in mixed batch samples of infected and healthy plants at a ratio of 1:50. The latter application could enable efficient screening of pepper plant nurseries or virus detection in the field. ELISA also could be used to detect PVY in potato (leaf and tuber), tomato, tobacco, Physalis floridana and Nicotiana glutinosa. Cucumber mosaic virus could be detected in all the above hosts, except potato, as well as in cucumber, celery, melon, squash and Datura stramonium. The relative titer of CMV or PVY in peppers could be evaluated by ELISA, which may become useful in breeding peppers by providing a quantitative evaluation of virus resistance.