Throat swabs from patients with pharyngitis and sputum specimens from patients with atypical pneumonia were tested for the presence of a Mycoplasma pneumoniae polypeptide with a molecular weight of 43,000 with the use of an M. pneumoniae species-specific monoclonal antibody in an immunoblot assay. This 43,000-dalton polypeptide was detectable in 33 of 33 throat swabs from patients with pharyngitis that were positive for M. pneumoniae by conventional culture as well as a culture-amplified enzyme immunoassay. The 43,000-dalton polypeptide was also detected in three of three M. pneumoniae culture-positive sputum specimens. It was not detected in 3 sputum specimens culture-confirmed for Legionella pneumophila, 10 sputum specimens from normal persons, or 25 throat swabs also from normal persons. This immunoblot assay could be completed within five hours and may be an alternative method for detecting M. pneumoniae antigen directly in sputum or throat swab specimens.