The utility of X-inactivation based clonality assays for evaluation of human neoplasia is well-documented. However, excessive Lyonization is a potential limitation of these assays, because it mimics clonal derivation of cells. The incidence of excessive Lyonization in healthy females is controversial, with reported incidence varying from 4% to 33%. Several explanations have been offered for the observed variation, including different criteria for excessive Lyonization, diversity of X- linked clonality assays, small population sizes and more recently, tissue specificity of X-inactivation patterns. However, it is also possible that stem cell depletion, clonal hematopoiesis, or selection pressures on blood cells results in an increased incidence of excessive skewing. If any of the latter is true, then the incidence of excessive skewing should increase with age in blood cells. To test this hypothesis, we determined X-inactivation ratios at the human androgen receptor locus of 295 normal females from three age groups: (1) neonates, (2) females 28 to 32 years old and, (3) females aged > or = 60 years. The incidence of skewing (allele ratios > or = 3:1) was 8.6% (14 of 162) in neonates (P = .104 v 28 to 32); 16.4% (11/67) in 28 to 32 y.o. (P = .0064 v > or = 60), and 37.9% (25 of 66) in women > or = 60 y.o. (P < .0001 v neonates). When a more stringent criterion for skewing was used (allele ratios > or = 10:1), the incidence was 1.9% (3 of 162) in neonates (P = .362 v 28 to 32), 4.5% (3 of 67) in 28 to 32 y.o. (P = .0022 v > or = 60), and 22.7% (15 of 66) in > or = 60 y.o. group (P < .0001 v neonates). Results have been confirmed at the phosphoglycerate kinase locus for 48 heterozygous females. The incidence of excessive skewing increases with age. In neonates, the incidence is low and may correspond to true excessive Lyonization. Acquired skewing occurs with aging in normal females and is present in 38% of females over the age of 60. Further studies are needed to determine whether acquired skewing is a consequence of stem cell depletion, true clonal hematopoiesis, growth advantage conferred by parental-specific X-chromosomes, or other causes. These data provide an explanation for variation in reported incidence of excessive skewing in normal females. Furthermore, these findings suggest that any study of clonality using X-inactivation based assays should incorporate age- matched controls for Lyonization.