Counterimmunoelectrophoresis (CIE) procedure has been established for mycoplasma identification from cultures, and the specificity and the sensitivity of this technique were evaluated. Preparations of milk samples and clinical and necropsy specimens were used for the technique. From 171 milk samples, 80 (46.8%) were positive by CIE (73 samples with Mycoplasma bovis and 7 with Mycoplasma arginini) and 77 (45%) were positive by biochemical and growth inhibition tests (70 samples with Mycoplasma bovis and 7 with Mycoplasma arginini). From 36 mycoplasma culture-positive clinical and necropsy specimens, CIE identified 5 different mycoplasma species and from 3 mycoplasma culture-positive specimens, the technique identified coinfection by 2 mycoplasma species. The CIE is a reliable, specific, sensitive, and rapid technique for the diagnosis of mycoplasmosis in the bovine.