This chapter provides an overview of tRNA, tRNA processing, and tRNA synthetases in Bacillus subtilis, emphasizing the areas in which there are distinct differences from Escherichia coli. It discusses the general characteristics of tRNA in the gram-positive bacteria. Despite the common belief that most bacteria are similar to E. coli, the tRNA gene organization and anticodon complement in B. subtilis, and probably in most gram-positive bacteria, are remarkably different from those in E. colii. The majority of the tRNA genes are in two large groups, one containing 21 and the other containing 16 tRNA genes, immediately following the 3′ ends of ribosomal gene operons. E. coli Ml-RNA has been shown to cleave modified tRNA-like substrates that consist of only the amino acid acceptor stem and the T-stem and loop. This explains how RNase P can recognize all of its tRNA precursor substrates and contrasts with the aminoacyl-tRNA synthetases that recognize many different nucleotide positions scattered throughout the tRNA. As aminoacyl-tRNA synthetases are so critical to the cell's metabolism, understanding their genetic regulation is especially important. Aminoacyl-tRNA synthetases have been divided into two classes on the basis of the presence of several different stretches of amino acid sequences. Class I synthetases include those for the amino acids arginine, glutamine, glutamate, isoleucine, leucine, methionine, tryptophan , tyrosine, and valine. Class II includes the aminoacyl-tRNA synthetases for aspartate, asparagine, histidine, lysine, phenylanine, proline, serine, and threonine.