The antigens of Mycoplasma hominis have previously been defined by means of functional assays with rabbit antisera and have been found to be predominantly protein. In this study, sera from humans as well as those from rabbits were shown to recognize protein antigens of M. hominis in an enzyme-linked immunosorbent assay (ELISA) that depended on antigen-binding capacity rather than biologic function. Although the ELISA was more independent of the strain of M. hominis used as antigen than was the mycoplasmacidal test, the use of no single strain allowed detection of more than 87% of positive sera; this observation suggests strain heterogeneity. In contrast to the results of other studies, common surface antigens among the strains of M. hominis used were demonstrated by immunoblot analysis of intact colonies.