Thiolactomycin inhibits bacterial cell growth through inhibition of the beta-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.