Normal Chinese hamster primary fibroblasts were exposed to methotrexate, an antifolate agent known to induce fragile sites. In a total of 156 banded metaphases prepared from methotrexate-treated cells, 93 breaks and gaps were observed and mapped to the individual hamster chromosomes and their bands. About two-thirds (69%) of all breaks and gaps mapped to the band Xq21, which is significantly more than expected. Some other bands exhibited fragility but the effect was not significant. In 6% of cells, homozygous fragility of Xq21 was observed, suggesting that this is a common fragile site. It has been reported previously that various chemicals induce preferential localized fragility of Xq in Chinese hamster ovary (CHO) and V79 cell lines. Our results suggest that preferential fragility of Xq in the presence of chemical compounds represents examples of fragile-site induction. A tendency of the in vitro chromosomal aberration test to yield positive results in the absence of positive data in other test systems, such as the Ames test and the mouse bone marrow micronucleus test, might be due to fragile-site expression induced by chemical compounds under study.