Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system. To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold. The activity of unmodified Phe-tRNA(Phe) on E. coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons. Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons.