Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. A recent structure-activity relationship study of 1 showed that replacing the C(6)-hydroxy group with alkoxy and thioalkoxy substituents led to dramatic losses of inhibitory activity in rho biochemical assays. The origin for this structural specificity has been explored by the synthesis and chemical, biochemical, and biological evaluation of C(6)-amino- (13), C(6)-(hydroxylamino)-(14), and C(6)-mercaptobicyclomycin (15). These compounds, like 1, are capable of entering into hydrogen bond donor interactions with rho and are capable of undergoing C(6) ring opening to generate alpha, beta-unsaturated carbonyl, imine, or thione systems. The chemical reactivity of 13-15 was compared with that of 1. We observed that 1, upon treatment with EtSH under moderate and basic conditions, readily underwent C(6) hemiaminal bond cleavage followed by conjugate addition to beta-methylene-alpha-ketoamide 2 to give Michael addition adducts whereas 13-15 reacted by initial cleavage of the C(1)-O(2) bond. Biochemical and biological assays of 13-15 and related analogues demonstrated that the C(6) hydroxy group in 1 was essential for activity. We found that replacing the C(6)-hydroxy group in 1 with weaker hydrogen bond donors led to low inhibitory activities in the rho-dependent ATPase and transcription termination assays. None of the bicyclomycin derivatives exhibited antibiotic activity against E. coli W3350 cells at a 32 mg/mL concentration. The apparent specificity for the C(6)-hydroxy group in 1 suggests that an efficient hydrogen bond donor interaction from the C(6)-hydroxy group to rho or the C(6) hemiaminal bond cleavage to 2 or both is necessary for drug function.