The precision of joint space width (JSW) measurements from plain film radiographs of the knee is limited by poor radio-anatomical positioning of the joint and/or insensitive methods of measurement. These limitations were overcome by establishing a precise radio-anatomical position for standing loaded and weight-bearing tunnel views of the knee, using the advantages of high definition macroradiography and a new computerized method for automatic JSW measurement from digitized macroradiographs of the knee. Reproducibility of JSW measurements was obtained from macroradiographs of knees of five post-mortem subjects and 12 patients with osteoarthritis (OA). JSW measurements were altered more by vertical than by horizontal misalignment of the X-ray beam relative to the joint space. In OA knees with medial compartment involvement, JSW measurements were more reproducible in the medial than lateral compartments in both radiographic views. In the medial compartment, the coefficient of variation for joint repositioning was 1% for minimum and 2% for average JSW, and for inter- and intraobserver errors, it was < 1% for both JSW measurements. The precision of this method will permit quantification of changes in JSW associated with disease progression and the treatment of OA.