The value of the hypervariable X-linked probe M27 beta for use in the analysis of X-chromosome inactivation patterns in normal blood and bone marrow cells and in the assessment of clonality in acute myeloid leukemia (AML) blast cells has been determined. By electrophoresing samples for 30 h, heterozygosity of the M27 beta locus was demonstrable in 324/415 females (78%) and this value could be increased to 93% by electrophoresing for 50 h. Determination of the X-chromosome inactivation patterns in blood and bone marrow samples from hematologically normal females was possible in approximately 90% of heterozygous individuals. The X-chromosome inactivation ratios obtained with M27 beta were comparable with phosphoglycerate kinase or hypoxanthine phosphoribosyl transferase in 46 individuals heterozygous for one of these genes in addition to M27 beta. The results obtained were closely correlated (r = 0.89). In 21 of 35 (60%) AML blasts, however, there was hypermethylation of the M27 beta locus and clonal analysis was not possible. Hypermethylation was not related to FAB type.