A new method of in vitro transcription with the use of SP6, T7, and T3 RNA polymerases is described. The method makes it possible to obtain, using only 1.6-2.6 micrograms of DNA template in as little as 50 microliters of transcription reaction in just 2 h, more than 200 micrograms of pure mRNA (with high translatability). Optimal conditions for the synthesis by all three RNA polymerases of transcripts 1500-1700 nt long and also for very long transcripts were determined. Reaction conditions that minimize DNA template or RNA polymerase requirements are also described, and these provide synthesis of either 1200-2100 RNA copies per DNA molecule or more than 5-15 micrograms of RNA per unit of RNA polymerase. Reactions can be easily scaled up to volumes of several milliliters, yielding up to 5.2 mg/ml of 1500- to 1700-nt-long RNA or up to 7.1 mg/ml of very long RNA.