Mycoplasma hyopneumoniae is the causal agent of Swine Enzootic Pneumonia, one of the most important diseases in the swine industry. Presently, the diagnosis ante-mortem is dificult because of the low sensibility and speciicity of the techniques currently in use. The purpose of this research was to standardize a nested Polymerase Chain Reaction (PCR) for the detection Mycoplasma hyopneumoniae in clinical samples from swine. For DNA extraction, different commercial and conventional methods were applied. The sensitivity 1 apulidov@unal.edu.co REV. MED. VET. ZOOT. 2006. 53:22-32 23 of the ampliication reaction was determined by using serial dilutions of the Mycoplasma hyopneumoniae Strain J, cultured in medium Friis. In addition, some modiications of the PCR reaction mixture were evaluated, and once the conditions were adjusted, the technique was applied to clinical samples such as nasal swabs and tracheobronchial washings. The best results were obtained when the bacterial DNA was extracted by heat, treated with Proteinase K and then used in PCR reactions without the Taq-polymerasa stabilizers, glycerol or BSA. Under these conditions, M. hyopneumoniae was detected up to 10-5 dilution of a pure culture of the reference Strain J, and also from several clinical samples.