The protective ability of the 3 mild isolates and 'Limiera' complex (control) of the Citrus tristeza virus (CTV) was evaluated on 23 seedless grapefruits by measuring yield and CTV visual symptoms, 14 years after performing the cross-protection experiment conducted in 1984, in Cordeirópolis, Sao Paulo, Brazil. An immunological diagnosis was carried out with different monoclonal antibodies and Single Strand Conformation Polymorphism (SSCP) of the virus coat protein gene to evaluate the correlation between the complexity of the CTV isolates of the plants and the field behaviour. The data related to stem-pitting symptomatology, yield, fruit size and vegetative development indicated a breakdown of the cross-protection with the 4 protective isolates. There was no correlation between virus titter and the severity of symptoms. The occurrence of mild and severe CTV isolates was demonstrated by serological evaluation. The SSCP pattern analysis showed the occurrence of more than 2 bands in every electrophoretic profile. Different banding patterns were observed among plants inoculated with the same isolate, indicating that different strain complexes were established during the experiment. Correlations among banding patterns and symptomatology suggested that some isolates may be used in future grapefruit cross-protection programmes.