Various ethyl and benzyl spermine analogues, including the anticancer agent N¹,N¹²-bis(ethyl)spermine, were studied for their ability to affect the growth of cultured Escherichia coli cells, to inhibit [³H]putrescine and [³H]spermine uptake into cells, and to modulate the peptidyltransferase activity (EC 2. 3. 2. 12). Relative to other cell lines, growth of E. coli was uniquely insensitive to these analogues. Nevertheless, these analogues conferred similar modulation of in vitro protein synthesis and inhibition of [³H]putrescine and [³H]spermine uptake, as is seen in other cell types. Thus, both ethyl and benzyl analogues of spermine not only promote the formation and stabilization of the initiator ribosomal ternary complex, but they also have a sparing effect on the Mg²⁺ requirements. Also, in a complete cell-free protein-synthesizing system, these analogues at low concentrations stimulated peptide bond formation, whereas at higher concentrations, they inhibited the reaction. The ranking order for stimulation of peptide-bond formation by the analogues was N⁴,N⁹-dibenzylspermine > N⁴,N⁹-bis(ethyl)spermine  N¹-ethylspermine > N¹,N¹²-bis(ethyl)spermine, whereas the order of analogue potency regarding the inhibitory effect was inverted, with inhibition constant values of 10, 3.1, 1.5, and 0.98 µM, respectively. Although the above analogues failed to interact with the putrescine-specific uptake system, they exhibited high affinity for the polyamine uptake system encoded by the potABCD operon. Despite this fact, none of the analogues could be internalized by the polyamine transport system, and therefore they could not influence the intracellular polyamine pools and growth of E. coli cells.