The present study aimed at characterizing the phenotype and functions of adherent synovial fluid (SF) cells derived from rheumatoid arthritis (RA), comparing with fibroblast-like synoviocytes (FLS) derived from RA synovial tissue (ST). Adherent SF-derived cells were spindle-shaped from passages 1-6 under light microscopy. The cell surface marker profile in SF-derived cells from passage 1-6 was similar to that of ST-derived FLS. Levels of MMP-1 and MMP-3 were not significantly different between SF-derived cells and ST-derived FLS (p = 0.20 and p = 0.40, respectively). There was no significant difference in the optical density value between two cell types in the cell invasion assay (p = 0.10). SF-derived adherent cells have a fibroblast-like phenotype from very early culture passages and have the potential to produce MMPs with the invasive capacity to degrade cartilage, identical to ST-derived FLS. Therefore, these cells could substitute for ST-derived FLS in studying the pathogenesis of RA.