We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN202-312). Tn5-induced hha mutants (hemolysin overproducers) segregated at high frequency either hemolysin-negative clones or clones showing the parental hemolytic phenotype (reduced hemolysin production). Secondary transpositions of Tn5 appeared to be responsible for the phenotypes of both types of derivatives. The hemolysin-negative clones no longer harboured Tn5 in the hha gene, but rather Tn5 was in the hly genes of the hemolytic plasmid pANN202-312. The derivatives that recovered the parental hemolytic phenotype contained a single copy of Tn5 in a chromosomal location different from that of hha. Both kinds of transpositional events appeared to restore the function of the hha gene.