Posttranscriptional modification in tRNA is known to play a multiplicity of functional roles, including maintenance of tertiary structure and cellular adaptation to environmental factors such as temperature. Nucleoside modification has been studied in unfractionated tRNA from three psychrophilic bacteria (ANT-300 and Vibrio sp. strains 5710 and 29-6) and one psychrotrophic bacterium (Lactobacillus bavaricus). Based on analysis of rural enzymatic hydrolysates by liquid chromatography-mass spectrometry, unprecedented low amounts of modification were found in the psychrophiles, particularly from the standpoint of structural diversity of modifications observed. Thirteen to 15 different forms of posttranscriptional modification were found in the psychrophiles, and 10 were found in L. bavaricus, compared with approximately 29 known to occur in bacterial mesophiles and 24 to 31 known to occur in the archaeal hyperthermophiles. The four most abundant modified nucleosides in tRNA from each organism were dihydrouridine, pseudouridine, 7-methylguanosine, and 5-methyluridine. The molar abundances of the latter three nucleosides were comparable to those found in tRNA from Escherichia coli. By contrast, the high levels of dihydrouridine observed in all three psychrophiles are unprecedented for any organism in any of the three phylogenetic domains. tRNA from these organisms contains 40 to 70% more dihydrouridine, on average, than that of the mesophile E. coli or the psychrotroph L. bavaricus. This finding supports the concept that a functional role for dihydrouridine is in maintenance of conformational flexibility of RNA, especially important to organisms growing under conditions where the dynamics of thermal motion are severely compromised. This is in contrast to the role of modifications contained in RNA from thermophiles, which is to reduce regional RNA flexibility and provide structural stability to RNA for adaptation to high temperature.