The study of fibroblast-like synoviocytes (FLS) has yielded important insights into the pathogenic mechanisms of rheumatoid arthritis. FLS can be cultured from synovial tissue obtained at joint replacement surgery, synovectomy, or synovial biopsy. After collagenase digestion, adherent cells consist mainly of synovial fibroblasts and synovial macrophages. Proliferating FLS are enriched by repeated passage and comprise >95% of cells by passage 3. Because of cell senescence, use of FLS lines after passage 9 is generally not recommended. FLS in culture have a distinct phenotype with regard to morphology, ultrastructure, surface phenotype, and function. Surface markers that can be used to characterize FLS include positive staining for VCAM-1, CD44, CD55, CD90 (Thy-1), and cadherin-11, coupled with the absence of macrophage markers such as CD14 or CD68.