In contrast to the well-characterized second messenger adenosine 3',5'-cyclic monophosphate (3',5'-cAMP), the biological roles of its isomer 2',3'-cAMP remain largely unknown, especially in bacteria. Recent work reported that RNase I-dependent elevation of 2',3'-cNMP levels in Escherichia coli correlated with reduced biofilm production, and separate studies demonstrated E. coli ribonuclease activation in response to aminoglycoside antibiotics. Here we report that E. coli produced 2',3'-cAMP in response to kanamycin at sub-inhibitory levels. Surprisingly, other aminoglycosides like streptomycin or gentamicin did not generate levels of 2',3'-cAMP detectable by P-31 NMR. Interestingly, because 2',3'-cAMP is also produced in E. coli strains expressing a plasmid-encoded kanamycin resistance gene but not by other ribosome-targeting antibiotics, this kanamycin-specific production may not reflect disrupted protein synthesis. Overall, this finding provides a link between aminoglycoside-induced ribonuclease activity and 2',3'-cAMP production in E. coli. (C) 2020 Elsevier Inc. All rights reserved.