Cysteinyl-tRNA synthetase (L-cysteine:tRNACys ligase (AMP-forming), EC 6.1.1.16) has been purified from rat liver in 23% overall yield. The enzyme was resolved by hydroxyapatite chromatography into three active forms (Fractions CRS-1, CRS-2 and CRS-3). The total activity ratio was about 0.7:2:1. The fractions CRS-2 and CRS-3 contained no other detectable aminoacyl-tRNA synthetase activity. CRS-2 was homogeneous by polyacrylamide gel electrophoresis, CRS-3 gave two active bands with mobilities corresponding to those of CRS-1 and CRS-2. The molecular weight of CRS-2 was about 240 000 by electrophoretic mobilities on the gels of various porosity, and 115 000-140 000 by sucrose gradient centrifugation. By gel-filtration, CRS-1, CRS-2 and CRS-3 exhibited apparent molecular weights of 122 000, 235 000 and 300 000, respectively. By sodium dodecyl sulfate gel electrophoresis, both CRS-2 and CRS-3 gave a single major band of 120 000 daltons. Stoichiometric study of cysteinyl adenylate formation indicated that CRS-2 has two active sites per molecule. These results are consistent with a dimeric structure of the type alpha2 for the major form of rat liver cysteinyl-tRNA synthetase, composed of two probably identical subunits of about 120 000 daltons. Available evidence also suggests that CRS-1 and CRS-3 are alpha and alpha3 (or alpha4), respectively.