Aim:This study was conducted to evaluate the in vitro effects of hydroxychloroquine (HCQ) on histone deacetylase (HDAC) enzyme activity and interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) expression. HDAC enzyme activity and the expression of inflammation markers were tested, with the presence of the HDAC inhibitor valproic acid, in human primary cell cultures prepared from two different tissues.Material and Methods:Primary cell cultures were prepared. Samples that did not receive any medication constituted the control group, while culture samples treated with HCQ served as the study group. The surface morphology of cells and the extracellular matrix (ECM) were evaluated by Giemsa staining and inverted light microscopy. Cell viability, proliferation, and cytotoxicity were determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium-bromide (MTT) analysis. The cultures were simultaneously stained with acridine orange (AO)/propidium iodide (PI) and viewed under fluorescence microscopy. HDAC enzyme activity and IL-6, IL-10, and TNF-α expression were evaluated using commercial enzyme-linked immunosorbent assay kits. The obtained data were analyzed using statistical methods. The alpha significance level was accepted as p < 0.05. Results:HCQ applied to cell cultures at the tested doses and durations showed cytotoxic effects on cell viability, proliferation, and cell or ECM morphology. It increased HDAC activity in chondrocytes and caused a proinflammatory response, indicated by an increase in TNF-α in the cells (p < 0.05). Conclusion:Taken together, the results emphasized that the cytotoxic effect of HCQ increased HDAC activity; therefore, this proinflammatory response should be taken into consideration in the clinical use of HCQ.