The use of D-lactic acid in differential diagnosis of bacterial arthritis was evaluated in a prospective study. The concentration of D-lactic acid was determined by the enzymatic UV-method in sixty-eight synovial fluids (SF) and in twenty four sera from adult patients with acute knee effusion. High concentrations of D-lactic acid (> 0.15 mmol/l) were measured most frequently in SF from bacterial arthritis, but also in individual culture-negative SF samples from patients with inflammatory culture-negative joint effusions with and without identified history of infections. Determination of SF D-lactic acid is not useful in differential diagnosis of bacterial arthritis.