Previous studies using hyperimmune antisera revealed significant serologic cross-reactions between Mycoplasma hyopneumoniae and M. flocculare. These findings, coupled with observations that M. flocculare infection may be widespread, indicated that antibodies to M. flocculare might be common in swine sera and thus cause nonspecific reactions in serodiagnostic tests for mycoplasmal pneumonia of swine (MPS). Information reported here indicates that this premise may be invalid. Twenty-one swine were infected experimentally with M. flocculare. Sera were collected every 2 weeks and tested against M. flocculare and M. hyopneumoniae antigens in an enzyme-linked immunosorbent assay (ELISA). Immunoassays against M. flocculare revealed that swine were slow to seroconvert, and that seroconversion was characterized by low titers. Significantly, none of the sera reacted with M. hyopneumoniae. M. hyopneumoniae reference antisera, and sera from swine naturally infected with M. hyopneumoniae and M. flocculare were also evaluated by an antibody inhibition procedure, i.e., sera were mixed with M. hyopneumoniae or M. flocculare antigen, incubated overnight and tested against M. hyopneumoniae antigen in the ELISA. Treatment with M. hyopneumoniae antigen markedly reduced or abolished seroreactivity, whereas treatment with M. flocculare antigen had little effect. These findings suggest that swine infected with M. flocculare usually do not develop sufficient levels of antibodies to detract from the specificity of the ELISA used to diagnose MPS.