An 18 A resolution structure of the 12-subunit yeast RNA polymerase II (RNAPII) calculated from electron microscope images of single particles preserved in amorphous ice reveals the conformation of the enzyme in solution. The Rpb4/Rpb7 polymerase subunit complex was localized and found to be ideally positioned to determine the path of the nascent RNA transcript. The RNAPII structure suggests a revised mode of interaction with promoter DNA and demonstrates that regulation of RNAPII must involve structural changes that render the enzyme competent for initiation.