Various authors have advocated quantitative methods of evaluating bone scintigrams to detect sacroiliitis, while others have not found them useful. Many explanations for this disagreement have been offered, including differences in the method of case selection, ethnicity, gender, and previous drug therapy. It would appear that one of the most important impediments to consistent results is the variability of selecting sacroiliac (SI) joint and reference regions of interest (ROIs). The effect of ROI selection would seem particularly important because of the normal variability of radioactivity within the reference regions that have been used (sacrum, spine, iliac wing) and the inhomogeneity of activity in the SI joints. We have investigated the effect of ROI selection, using five different methods representative of, though not necessarily identical to, those found in the literature. Each method produced unique mean indices that were different for patients with ankylosing spondylitis (AS) and controls. The method of Ayres (19) proved superior (largest mean difference, smallest variance), but none worked well as a diagnostic tool because of substantial overlap of the distributions of indices of patient and control groups. We conclude that ROI selection is important in determining results, and quantitative scintigraphic methods in general are not effective tools for diagnosing AS. Among the possible factors limiting success, difficulty in selecting a stable reference area seems of particular importance.