In 1981, a recurrent local burst of high mutability and high allele frequency of the yellow gene was recorded in the natural population of Drosophila melanogaster from Uman', Ukraine. A detailed genetic analysis showed that hypomorphic alleles y2 prevailed during the mutation burst. Mutations were strictly allele-specific and occurred only in two directions: from y2 to y+ and from y+ to y2. Alleles y2 isolated from the natural population differed in the degree of instability of mutant and wild-type derivatives, expression of the mutant phenotype, and complementation properties. In this work, the insertion nature of all unstable alleles derived from the natural population is confirmed by direct molecular methods. The mutation burst at the yellow locus is shown to result from insertion of a defective hobo copy. Six mutations y2 independently isolated from the natural population were caused by an insertion of hobo in the same site of the yellow regulatory region. The hobo copies were identical according to the restriction map. In three y2 alleles, the mutant phenotype was associated with inversion between hobo copies, one of which was located in the yellow locus and the other, in another unidentified region of the X chromosome. The remaining y2 alleles were associated with deletions that were located in the vicinity of the hobo insertion site and in the region of yellow enhancers.