We describe herein, a new unstable mutant of the vestigial locus, isolated from a French natural population. From this mutant vestigial almost (vgal) wild-type flies (vgal+) and extreme vg phenotypes (vge) arose spontaneously without genomic shock. The occurrence of vgal+ or vge alleles depends mostly on the breeding temperature; vgal+ revertants arose principally at low temperature (21 degrees C) and vge at 28 degrees C. These events occur mainly in the male germ line and the phenomenon appears to be premeiotic. Our results with in situ hybridization experiments and Southern blots show that the vgal mutation is due to a 2 kb DNA insertion, which is a deleted hobo element. Genetic and molecular analyses show that two distinct events may underly the wild-type revertants. One is the excision of the resident hobo element, the other a further deletion (about 300 bp in the example characterized herein). The vge mutation is probably due to a deletion of vestigial sequences flanking the hobo insertion.