We applied a real-time quantitative assay to determine the expression of tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) in tissue samples. This method is based on the real-time monitoring of reverse transcriptase-polymerase chain reaction (RT-PCR) with a dual-fluorescence-labeled probe and a sequence detector. A linear correlation existed between assay measurements and input target (r = 0.999). TNF-alpha mRNA was detected in the synovial cells of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and also in the U937, THP1, and HL60 cell lines. Stimulation with interleukin-1alpha (IL-1alpha) caused an immediate increase in the intracellular level of TNF-alpha mRNA. In particular, the relative copy number of TNF-alpha mRNA increased dramatically from 15 to 3554 in synovial cells from RA patients. This method is simple, accurate, and sensitive for the quantitative detection of TNF-alpha mRNA. The real-time quantitative RT-PCR assay may be valuable for measuring TNF-alpha mRNA expression in clinical samples.