Tn4560 (8.6 kb) was derived from Tn4556, a Tn3-like element from Streptomyces fradiae. It contains a viomycin resistance gene that has not been used previously for selection in mycobacteria. Tn4560, cloned in a Streptomyces plasmid, was introduced by electroporation into Mycobacterium smegmatis mc(2)155. Tn4560 transposed into the host genome: there was no obvious target sequence preference, and insertions were in or near several conserved open reading frames. The insertions were located far apart on different AseI macrorestriction fragments. Unexpectedly, the transposon delivery plasmid, pUC1169, derived from the Streptomyces multicopy plasmid pIJ101, replicated partially in M. smegmatis, but was lost spontaneously during subculture. Replication of pUC1169 probably contributed to the relatively high efficiency of Tn4560 delivery: up to 28% of the potential M. smegmatis transformants acquired a stable transposon insertion. The data indicated that Tn4560 may be useful for random mutagenesis of M. smegmatis.