In vitro and in vivo immunization techniques were applied for the production of monoclonal antibodies against the aster yellows (AY) agent, a phytoplasma. The immunogens purified from diseased lettuce using discontinuous Percoll density gradients, when compared with those directly extracted from the salivary glands of infective insect vectors applied in the previous study, enhanced the proportion of AY-specific antibody-producing hybridomas in the total antibody-secreting hybridoma populations in both in vitro and in vivo trials. The in vitro immunization, when compared with in vivo immunization, yielded an even higher proportion of AV-specific antibody-producing hybridomas. Sixteen stable hybridoma cell lines were produced, each secreting specific monoclonal antibodies against the AY agent . When using indirect biotinylated enzyme -linked immunosorbent assay (ELISA) , these monoclonal antibodies reacted specifically to the AY-infected plants and differentiated the AY agent from other phytoplasmas associated with seven other diseases.