We have used two-dimensional polyacrylamide gel electrophoresis to fractionate tRNAs from Escherichia coli. A sufficiently high degree of resolution was obtained for 44 out of 46 tRNA species in E. coli to be resolved into individual electrophoretic components. These isolated components were identified by hybridization to tRNA-specific oligonucleotide probes. Systematic measurements of the abundance of each individual tRNA isoacceptor in E. coli, grown at rates varying from 0.4 to 2.5 doublings per hour, were made with the aid of this electrophoretic protocol. We find that there is a biased distribution of the tRNA abundance at all growth rates, and that this can be roughly correlated with the values of codon frequencies in the mRNA pools calculated for bacteria growing at different rates. The tRNA species cognate to abundant codons increase in concentration as the growth rate increases but not as dramatically as might be anticipated. The levels of most of the tRNA isoacceptors cognate to less abundant codons remain unchanged with increasing growth rates. The result of these changes in tRNA abundance is that the relative increase in the amounts of major tRNA species in the bacteria growing at the fastest growth rates is more modest than previous estimates from this laboratory suggested. Furthermore, a systematic error in previous estimates of ribosomal RNA content of the bacteria has been detected. This will account for the quantitative discrepancies between the previous and the present data for tRNA abundance.