In the attempt to further characterize the extent and timing of tumor necrosis factor (TNF)alpha-system activation during multiple sclerosis (MS), we performed a cross-sectional and a longitudinal study in a total of 73 relapsing-remitting MS patients. We assessed serum levels of soluble TNFalpha, soluble TNFalpha receptor 1 (R1) and soluble TNFalpha receptor 2 (R2) in 65 relapsing-remitting MS patients in different phases of disease. TNFalpha, R1 and R2 serum levels measured in MS patients did not differ from those measured in healthy individuals and did not correlate with (a) clinical relapses, (b) presence of gadolinium-enhancing brain-magnetic resonance imaging (MRI) lesions, and (c) bioactivity of TNFalpha. We also measured in 8 additional relapsing-remitting MS patients peripheral blood mononuclear cells (PBMC) mRNA levels of TNFalpha, R1 and R2 every 15 days for one year. In 4 of these patients we also measured levels of soluble TNFalpha, R1 and R2 every 15 days for 5 months across a clinical exacerbation. PBMC TNFalpha, R1 and R2 mRNA levels and serum levels of soluble R1 and R2, but not TNFalpha, fluctuated concordantly (P<0.05) and peaked a mean of 6 weeks before clinical and MRI evidence of disease activity. Moreover, we found a significant positive correlation between cumulative TNFalpha and R2 mRNA levels (measured during the follow-up period in the 8 MS patients studied serially) and the number of clinical attacks recorded in these patients during the study. Our data show that serum levels of soluble TNFalpha, R1, and R2 in MS patients do not differ from those of healthy individuals. However, although within normal values, the transcription and production rate of all these molecules fluctuate concordantly in the peripheral blood during the course of the disease (with the exception of soluble TNFalpha) and their maximal elevation significantly precedes the occurrence of clinical exacerbations. It is not clear whether soluble TNFalpha escapes recognition by commonly used assays or is simply not released in its soluble form in MS patients. In any case, measurement of TNFalpha mRNA levels and R1 and R2 mRNA and protein levels appears to be a better indicator of disease fluctuations during the course of MS than assessments of soluble TNFalpha protein.