OBJECTIVE (1) To define the optimal conditions for transwell assay of synoviocyte chemotaxis. (2) To develop a live cell imaging chemotaxis assay for synoviocytes. (3) To characterize the chemotaxis of rheumatoid synoviocytes (RAS). RESULTS Optimal conditions for transwell assay of synoviocyte chemotaxis were 8 microns pore size filters coated with collagen I (C1), assayed for 24 hours. Without the C1 coating 2.9 x 10(3) RAS migrated to the lower chamber. This increased to 4.7 x 10(3) when 20 micrograms/ml fibronectin (Fn) was added. With the C1 coating 4.3 x 10(3) cells migrated through the filter without chemotactic stimulation compared to 12.8 x 10(3) with interleukin 1 beta (IL-1 beta) 5 ng/ml, 12.2 x 10(3) with granulocyte-macrophage colony stimulating factor (GM-CSF) 25 ng/ml, 11.7 x 10(3) with Fn 20 micrograms/ml, and 9.0 x 10(3) with transforming growth factor-beta 1 (TGF-beta 1) 20 ng/ml (all p < 0.01). In the imaging assay, 50.7% of the RAS migrated toward the C1 coating without bound chemoattract. The percentage of cells migrating toward each chemoattractant at its optimal concentration was 64.3% for IL-1 beta, 60.8% for IL-8, 64.7% for GM-CSF, 61.0% for Fn, 58.9% for IL-6, and 69.1% for TGF-beta 1 (all p < 0.01). All of these chemoattractants increased directed migration without changing the random migration. Indomethacin (100 ng/ml) and Dexamethasone (10 ng/ml) inhibited Fn-induced chemotaxis. CONCLUSIONS We report two in vitro assays for synoviocyte chemotaxis adapted and optimized for the study of synoviocytes. The live cell imaging assay had the advantage that it could separate directed and random migration.