Palindromic or semipalindromic sequences can adopt cruciform structures in DNA in vitro. It has been demonstrated in some cases that A/T-rich cruciforms exist also in vivo in Escherichia coli. The biological function of those structures is not understood although putative cruciforms have been found in interesting locations on replication origins, operators, or transcriptional termination regions. Here we show by means of the use of structure-dependent nucleases that the 3' end of the yeast FBP1 gene contains a stable cruciform both in vitro and in E. coli cells and that in both cases, its extrusion depends on the DNA supercoiling state. © 1993 Academic Press, Inc.