This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, Bg/I, Bg/II, Hind II, HindIII, HpaI, Sa/I, Aval, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColEl::Tn5 plasmids, and a ColEl::Tn5 deletion derivative. Ba/I, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColEl::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a Bg/II site within this segment results in loss of the neomycin resistance phenotype. Since this Bg/II site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.