Expression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(p12), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did not induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra(16)(p12) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.