The histidyl-tRNA synthetase of rabbit reticulocyte cytosol has been purified 84 000-fold to apparent homogeneity with a specific activity of 687 nmol of histidyl-tRNA formed per min per mg of protein. Ten to 15% of the enzyme activity is sedimented with the ribosomes while the remainder is in the cytosol. The purified enzyme has a molecular weight of 122 000 as determined by sucrose density gradient centrifugation. Gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate suggests that it is composed of two similar subunits with a molecular weight of approximately 64 000. The enzyme has a magnesium optimum of 45 mM; however, this is reduced to 5 mM in the presence of an intracellular potassium concentration (160 nM). The enzyme acylates the two histidine tRNA isoacceptors of rabbit reticulocytes with similar Km values and at similar rates.