The sex-chromosomal origin of the ring chromosome in a pre-pubertal non-virilized female patient presenting with a 45,X/46,X,r(?) karyotype could not be resolved by conventional cytogenetic (including G11) methods. Non-autoradiographic in situ hybridization of biotinylated X and Y centromere-specific alphoid repetitive sequence probes unequivocally and rapidly identified the ring to be of X origin.