Naturally occurring seleno-tRNAs from Methanococcus vannielii account for 13-20% of the total tRNA population. Two different selenium-modified nucleosides were detected in these seleno-tRNAs. Of the total selenium incorporated, about 60% is present in 5-methylaminomethyl-2-selenouridine, and the other 40% occurs in a second selenonucleoside with spectral characteristics typical of a derivative of 2-selenouridine. The 5-methylaminomethyl-2-selenouridine was found in the seleno-tRNA species present in the early fractions of a linear salt gradient elution profile from a reversed-phase chromatographic system 5 (RPC-5) column, whereas the second selenonucleoside occurred in the tRNA species eluted late in the profile.