The parental origin and mechanism of formation of polysomy X were studied in two polysomic cases, using four X-linked restriction fragment length polymorphisms, three (CA)n dinucleotide repeat sequences and one variable number tandem repeat (VNTR) locus as genetic markers. A nonradioactive technique based on the hybridization of the polymerase chain reaction (PCR) product was developed for the analysis of dinucleotide repeats. Segregation analysis using different nonradioactive approaches based on the PCR, revealed that all four X chromosomes were of maternal origin. These data provide additional evidence of an identical mechanism of successive nondisjunctions in maternal meiosis I and II.