An immunobinding assay was developed to identify mycoplasma colonies on agar in pure and mixed cultures. Mycoplasma colonies on agar were transferred to nitrocellulose. The nitrocellulose was treated with specific rabbit antisera against mycoplasmas, peroxidase-conjugated goat anti-rabbit immunoglobulin G, 4-chloro-1-naphthol, and H2O2. Purple developed in the presence of specific reactions. The procedure was superior to epifluorescence.