Turner syndrome is caused by haploinsufficiency of the short arm of X-chromosome, and is usually diagnosed by karyotyping. This procedure is time-consuming, expensive and unfeasible for population screening. We propose molecular detection of 45XO Turner patients based on the ability of HpaII, a methylation sensitive endonuclease, to induce the cleavage of non-methylated DNA in the active X-allele. Genomic DNA was obtained from 22 patients with Turner syndrome confirmed by karyotype (45XO, N = 18; 45XO/46XX, N = 4). After digestion, DNA was amplified with primers directed to exon 1 of the androgen receptor (AR) gene and to the GAPDH control gene. Normal control females or mosaic patients, with a second methylated X-chromosome, escaped from HpaII digestion and produced a band corresponding to AR gene amplification. 45XO patients have just one active non-methylated X-chromosome, completely digested by HpaII, thus preventing the amplification of the AR gene. Three of the 45XO cases gave amplified bands, suggesting low-frequency mosaicisms that are not detected by karyotyping. Compared to classical karyotype studies for the detection of 45XO Turner patients, this new molecular method is simpler, faster and less expensive.