The sigma subunit of Escherichia coli RNA polymerase was released from a transcribing polymerase-poly[d(A-T)]-poly[d(A-T)]-RNA complex when the nascent RNA reached a length of either eight or nine nucleotides. The dissociated sigma was separated from other reaction components by gel filtration, and was assayed using an activity assay previously described (Hansen, U. M., and McClure, W. R. (1979) J. Biol. Chem. 254, 5713-5717). The extent of RNA synthesis was limited by incorporation of 3'-dATP into the RNA chains; a series of distributions of product lengths was achieved by varying the concentration of 3'-dATP. A correlation of the number of moles of dissociated sigma versus the number of moles of RNA products of varying lengths allowed the determination of the point of release of the sigma subunit. Models to explain the cause of sigma release are discussed.