Examination of sputum specimens can be used for monitoring airway inflammation by means of immunological and molecular techniques. RNA extraction is essential for measurement of cytokine gene expression and for detection of many viral pathogens in sputum. In this study, three RNA extraction methods used commonly (acid guanidinium thiocyanate-phenol-chloroform extraction, Trizol and RNeasy Mini kit) were compared on the sputum of 14 patients who had chronic obstructive pulmonary disease. The effect of dithiothreitol pre-treatment on sputum RNA extraction was also investigated. The yield and purity of total RNA were determined by spectrophotometry. Reverse transcription-polymerase chain reaction (RT-PCR) results of the house keeping gene (glyceraldehyde-3-phosphate dehydrogenase) and RNA sequences specific to common respiratory viruses were compared. The results showed that (1) total RNA extracted with Trizol had highest yield and purity among the three RNA extraction methods; (2) there was no significant difference among the three RNA extraction methods on the house keeping gene and viral detection by RT-PCR; (3) dithiothreitol pre-treatment did not improve either the purity of total RNA, or RT-PCR signal. Moreover, dithiothreitol treatment reduced significantly the yield of total RNA. The results of the study indicate that the Trizol method appears to be superior for total RNA extraction from sputum, and dithiothreitol pre-treatment does not increase the efficiency of RNA extraction and RNA detection in sputum specimens.