ObjectiveTo ascertain the importance of alignment of the medial tibial plateau (MTP), as determined by the distance between the anterior and posterior margins of the plateau (intermargin distance [IMD]), for measurements of joint space width (JSW) in radiographs of normal knees. MethodsJSW and IMD were measured in paired baseline and 12-month knee films of 122 subjects from the osteoarthritis initiative (OAI). Relationships between JSW and IMD, and between the variation in JSW and variation in IMD, were evaluated. ResultsIn cross-sectional analysis, a non-linear relationship existed between JSW and the concurrent IMD. With poor MTP alignment (IMD>1.7mm), a 1.0-mm increase in IMD resulted in a 0.16-mm (95%CI: 0.11–0.21) increase in JSW (P<0.0001). In a longitudinal analysis, the effect of IMD variation on variation in JSW was also highly significant (P<0.0001). A variation of 1mm between IMDBaseline and IMD12month was associated with a 0.10-mm (95% CI: 0.06–0.13) variation in JSW, with variations in JSW and IMD occurring in the same direction. An IMD variation less than or equal to 1.0mm was determined to be acceptable for accurate evaluation of JSW in serial radiographs. ConclusionThe error in measurement of JSW caused by variation in IMD in serial radiographs of normal knees can be as large, or larger, than the mean rate of 12-month joint space narrowing (JSN) in OA knees. MTP alignment and replication of alignment in serial knee films are required for accurate determination of JSN in OA knee.