Expression of fragile X [fra(X)] (q27.3) and endoreduplicated metaphases have been reported in methotrexate-treated (MTX) fra(X) cultures (Kerem B, Biotein R, Schaap T : Chromosoma 97: 6-10). Further, new data (Kimchi-Sarfaty C, Goitein R, Kerem B, Werner M, Medan B, Schaap T : Am J Med Genet, this issue) indicate that MTX may specifically induce polyploidy and endoreduplication in cells with the fra(X) mutation. To confirm and extend these results, we have studied short-term lymphocyte cultures incubated in M199, a folate deficient system, and RPMI-1640 in the presence and absence of 5-fluorodeoxyuridine (FUdR) exposure during the last day of a 4 day culture. No endoreduplicated cells were seen under these conditions and there was no change in the level of polyploidy. We also studied the distribution of polyploid and endoreduplicated cells in amniotic fluid and chorionic villus sample cultures from one fra(X) positive and 4 at-risk specimens. No increase in the incidence of polyploidy or endoreduplication was observed in cultures exposed to MTX for both 24 and 48 hours from a fra(X) positive amniotic fluid case. Cytogenetic results were fra(X) negative for the remaining 4 cases tested. There was significant discordance between our findings and those expected based on MTX-induced increased frequencies of polyploidy and endoreduplication. Thus, our studies do not confirm the reported correlation between the presence of FRAXA and increased frequencies of polyploidy and endoreduplication in MTX-exposed amniocyte cultures and there was no evidence for increased levels of polyploidy and endoreduplication in short-term fra(X) lymphocyte cultures exposed to non-MTX fra(X) induction.