Nasopharyngeal aspirates and throat swab specimens were compared in a polymerase chain reaction (PCR)-based test for Mycoplasma pneumoniae. The pathogen was detected in 50% and 45% of throat swab specimens and aspirates, respectively. However, in specimens negative for Mycoplasma pneumoniae by PCR, amplification inhibitors were detected in 0% and 36% of throat specimens and aspirates, respectively. Further investigations confirmed that no throat specimens, but one-quarter of aspirates, are likely to be rejected for containing inadequate respiratory material or excess amplification inhibitors. Because rejection of most of the unsuitable specimens is possible only after PCR, the use of aspirates is less cost-effective. This, and the reluctance to subject patients to aspiration, make the aspirate an inferior specimen for detection of Mycoplasma pneumoniae by PCR.