Recently, we demonstrated that early low concentrations of circulating, adalimumab-bound TNF in RA patients treated with adalimumab was associated with future anti-drug antibody formation. Furthermore, low TNF was associated with less frequent baseline MTX use. This is remarkable, because of the anti-inflammatory effects of MTX and a potential inhibiting effect on cytokine production. We hypothesized an indirect effect of non-MTX use on low TNF concentrations via immunogenicity. To investigate the effect of MTX on TNF concentrations independent of anti-drug antibody formation, we measured TNF in RA patients treated with etanercept, a drug with low immunogenicity. Methods: TNF was quantified in 186 consecutive etanercept-treated RA patients at baseline and at weeks 4, 16 and 28. The dynamics of TNF during etanercept treatment were compared with dynamics recently published for adalimumab. Results: We demonstrated that TNF concentrations at week 4 did not associate with baseline MTX or remission after 28 weeks. Furthermore, median (interquartile range) TNF increased from <112 (<112-<112) pg/ml at baseline to 548 (344-688) pg/ml at week 4 and remained stable at week 16 and 28 [598 (442-756) and 568 (444-755) pg/ml, respectively]. Conclusion: Circulating TNF did not associate with MTX usage in etanercept-treated patients. This implies that MTX does not have a direct effect on TNF concentrations in circulation and that the association between early low TNF and non-use of MTX for adalimumab is thus most likely due to anti-drug antibody formation.