Twenty Escherichia coli RNA polymerase molecules bind specifically to linear, double-stranded coliphage λ DNA at 30:1 polymerase-to-λ DNA molar ratio, under the conditions used. The binding sites were identified by electron microscopy employing a glutaraldehyde/BAC technique which measures binding with 84% specificity. Binding sites could be assigned to all well identified λ promoters, including p I, p L, p rm, p r, p o and p r, although only one bound polymerase could be found in the p rm− p R region, probably reflecting the low affinity of RNA polymerase for the P rm promoter. Several binding sites seem to correspond to minor in vivo-active transcriptional startpoints (e.g., of lit or mis RNA), to potential promoter sites (e.g., hip), or to the startpoints observed only during in vitro enzymatic RNA synthesis (e.g., the b2 region and the 96 to 99.5% λ region). Moreover, a few binding sites are in the regions that bear no known startpoints but contain known transcriptional terminators. Correlation between the efficiency of initiation of RNA synthesis and the frequency of RNA polymerase binding is good only for some promoters. All of the RNA polymerase binding sites lie within the A + T-rich regions, as determined by partial denaturation mapping. However, quantitative correlation between frequencies of polymerase binding and localized DNA melting is far from perfect.