The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer with each subunit having a complete active site. The enzyme-substrate complexes, however, are known to be asymmetrical in solution because the enzyme exhibits half-of-the-sites activity by binding tightly only 1 mol of tyrosine or 1 mol of tyrosyl adenylate per mole of dimer. Evidence is now presented that the unligated enzyme is also asymmetrical in solution. Symmetry was investigated by construction of heterodimers containing one full-length subunit and one truncated subunit, allowing the introduction of different mutations into each monomer. Each dimer is active at only one site, but the site used is randomly distributed between the subunits. Each heterodimer thus consists of two equal populations, one activating tyrosine at a full-length subunit and the other at the truncated subunit. No detectable interconversion is found between active and inactive sites over several minutes either in the absence of substrates or when the enzyme is turning over in the steady state. Kinetic evidence implies that wild-type enzyme is inherently asymmetrical even in the absence of substrate.