An alphoid DNA sequence primarily located on the X chromosome was labeled with biotin and hybridized in situ to preparations of metaphase chromosomes derived from fragile X-affected individuals; hybridization sites were detected immunologically. Labeled X chromosomes were located automatically in digitized images of metaphase cells by searching for the concurrence of a pronounced peak in the longitudinal density profile with the centromere in medium-sized chromosomes having a suitable centromeric index. Approximately 70% of the X chromosomes were detected by a simple classifier; this rate is similar to the automatic classification rate obtained with G-banded metaphases. The frequency of detection of the fragile X site obtained when scored directly from the microscope using this new preparation technique did not differ significantly from the frequency obtained in the same sample by means of a conventional technique. The frequency obtained by visual scoring of digitized images was slightly higher, but not significantly so.