The distal long arm of the X chromosome contains at least 2 fragile sites, the well known rare site at Xq27.3 (FRAXA), associated with the fragile-X syndrome, and a common fragile site at Xq27.2 (FRAXD), inducible by high doses of aphidicolin. Lesions at Xq26 have also been reported in lymphocytes of mentally retarded individuals cultured under folate deprivation or thymidylate stress. This study determines the frequency of the fragile site at Xq27.2 and lesions at Xq26 in individuals referred to our laboratory to rule out the fragile-X syndrome and in control individuals using our routine culture system for the diagnosis of the syndrome. FRAXD was expressed in 1/20 (5%) individuals in each of the study groups, in 1-2% of cells. Lesions at Xq26 were found in 1-2% of the lymphocytes of 5/166 (3%) patients referred for fragile-X analysis who were FRAXA negative, and in 1% of cells of 1/20 (5%) control individuals. We conclude (1) the fragile site at Xq27.2 can be demonstrated in normal individuals under conditions of thymidylate stress routinely used for cytogenetic diagnosis of the fragile-X syndrome, (2) this fragile site is present at low levels (1-2%) in all individuals who express it and, therefore, its expression is unlikely to cause false positive diagnoses of the syndrome, (3) lesions at Xq26 are also seen at low levels in lymphocytes of individuals without the syndrome, and (4) accurate differentiation of the rare site at Xq27.3 from other distal Xq fragile sites or lesions will lead to avoidance of unnecessary repeat studies.