Aminoacylation of tRNA occurs with a high degree of accuracy in many cases because misacylated molecules are effectively proofread on the aminoacyl-tRNA synthetase by preferential hydrolysis. This hydrolysis releases an ATP equivalent of energy. An explicit relationship between cost of proofreading and the resulting degree of accuracy is presented. Experimental data from Escherichia coli for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle are examined by means of this relationship, and a conjecture concerning the natural selection of accuracy and proofreading costs is put forth and tested. We have found the energy cost of accurate proofreading to be high. The minimum error, derived in previous theoretical studies, is never actually reached. Instead, higher values, determined by the balance between energy wasted in the cell as a consequence of error and the energy cost of proofreading, appear to be selected. The total cost of proofreading all types of tRNA aminoacylations is estimated to be approximately 2% of the energy required to synthesize a bacterial cell.