The L-lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae, formerly named protein P36, belongs to the predominant immunogenic proteins in pigs which were naturally or experimentally infected with M. hyopneumoniae. The antigenic reaction against M. hyopneumoniae LDH has been shown to be species specific. Recombinant M. hyopneumoniae LDH, which was genetically engineered to contain six histidine residues at its C-terminal end, was expressed in E. coli and purified to a high degree using Ni-chelate affinity chromatography. The genetically engineered LDH still showed the same biochemical activity and immunological specificity as the wild-type LDH and was used as an antigen for a M. hyopneumoniae LDH ELISA. Using this assay, we showed that pigs experimentally infected with M. hyopneumoniae raised antibodies against LDH in two steps. An early, relatively weak anti-LDH response was detected between 5 to 10 weeks post-infection when clinical signs and lung lesions occur. This first minor raise of anti-LDH antibodies occurred simultaneously with the strong appearance of antibodies against an antigen consisting of membrane proteins of M. hyopneumoniae prepared with Tween 20 extraction. A second, strong raise in anti-LDH antibodies was observed from the twelfth week after infection, at a time when the disease signs and the infectious agent disappeared. The high anti-LDH titer persisted until 21 weeks post-infection, in contrast to the antibody titer against the membrane proteins which started to decrease after its peak at 12 weeks post-infection. A LDH-ELISA may also be useful for detecting past infections.