The variation among natural isolates of tomato spotted wilt virus (TSWV) from France, Belgium, and Italy was studied. These isolates could be classified into three groups according to their symptomatology in cowpea. However, they did not show any serological variation with polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) raised against the Brazilian TSWV isolate BR-01. The effect of repeated mechanical transfers of three isolates representing each cowpea symptom group, LAS 6329, LYE 51, and LYE 52, on symptomatological and serological properties was investigated. After six serial passages in tobacco, LYE 51 did not cause symptoms in lettuce or tomato plants. After 14 passages in tobacco, the symptoms caused by LAS 6329 and LYE 52 on a limited host range, which included cowpea, lettuce, tobacco, and tomato, were similar to those caused by the original isolates. Leaf extracts from lettuce and tomato, inoculated after 14 passages in tobacco, gave similar enzyme-linked immunosorbent assay (ELISA) values with PAbs and MAbs directed to the nucleocapsid (N) protein. MAbs raised against glycoprotein reacted positively in ELISA only with extracts of tomato plants; the reaction with extracts of lettuce inoculated with LYE 52 following serial passage through tobacco was comparable with the background readings obtained with extracts of healthy lettuce plants. When serial transfers were made in lettuce and tomato, ELISA values were reduced in both hosts compared with the ELISA value of the original isolate in tomato.