AbstractTo evaluate the suggested nonrandom folding of Xql3‐q21 (center of Barr body condensation) of the inactivated X chromosomes, metaphases from nine subjects with or without X chromosome abnormalities (eight females and one male) were investigated with RBG‐staining. A significant increase (p < .05) in the number of arm folds (Xq13‐q21) of the late‐replicating X chromosome, particularly in early to mid‐metaphase, was observed in four of eight females. Therefore, the stage of chromosome contraction was an important factor with more folds observed at the centromeres and in longer chromosomes in early metaphase compared with mid‐ to late metaphase. X chromosome folds were present in cells of subjects treated with or without bromodeoxyuridine. While our study agrees with the relationship of Xql3‐q21 fold with the X‐inactivation center, the correlation of extended chromosomes and folding limits this method as a sole indicator of X‐inactivation in routine mid‐metaphase, but is useful in the analysis of early metaphase chromosomes.