Early diagnosis of Mycoplasma pneumoniae (Mp) plays a pivotal role in its management. We evaluated the role of rapid culture method in early diagnosis of Mp infection and discussed the potential impact factors. A total of 2,600 patients with acute respiratory infection were included, and their pharyngeal swab samples were prepared for Mp rapid culture based on selective Mp fluid culture medium. The clinical contributing factors related to Mp infection were also explored. The positive rate of Mp culture in females was 41.75%, which was higher than that for males (37.63%). Mp infections were incidental to the children and elderly. The positive rates of Mp culture were higher in children aged 3-5 years and adults older than 70 years (54.05 and 31.48%, respectively), compared with other ages. In addition, Mp infection frequently occurred in winter (December February) and spring (March May), with significantly higher positive rates of Mp culture by 40.02 and 42.89 vs. 32.15 and 33.50% in summer (June August) and autumn (September November), respectively. The positive rate of rapid culture for Mp was slightly higher than that by Mp antibody assay, but the diagnostic accordance was well between these two methods (P>0.05). Furthermore, clinical common symptoms of respiratory tract infection, such as fever, cough, and expectoration, were not found specific in Mp infection, suggesting that they were not independent prognostic predictors for Mp infection. Therefore, rapid culture based on the Mp pathogen detection would have important clinical application for the early diagnosis of Mp infection. J. Clin. Lab. Anal. 24:224-229, (C) 2010 Wiley-Liss, Inc.