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The causative agent of porcine mycoplasmal pneumonia, Mycoplasma hyopneumoniae, is difficult and time-consuming to isolate. Serological identification using antibodies induced by the disease is confused by cross-reaction with a closely related organism,Myc. flocculare. From pig lungs obtained at slaughter for meat and deemed free of acute disease, it was possible to detect by culture both Myc. hyopneumoniae and Myc. flocculare. This study has improved on an earlier PCR detection of DNA from the former species by using a nested PCR capable of detecting the purified DNA equivalent to one mycoplasmal genome. With this PCR assay both mycoplasma species were detected and differentiated directly from lung tissue.