Glutamyl-tRNA reductase catalyzes the initial step of tetrapyrrole biosynthesis in plants and prokaryotes. Recombinant Escherichia coli glutamyl-tRNA reductase was purified to apparent homogeneity from an overproducing E coli strain by a two-step procedure yielding 5.6 mg of enzyme per gram of wet cells with a specific activity of 0.47 mumol min-l mg(-1). After recombinant production, denatured glutamyl-tRNA reductase from inclusion bodies was renatured by an on-column refolding procedure. Residual protein aggregates were removed using Superdex 200 gel-filtration chromatography. Solubility, specific activity, and long-term storage properties were improved compared to previous protocols. Obtained enzyme amounts of high purity now allow the research on the recognition mechanism of tRNA(Glu) and high-throughput inhibitor screening.