Purified calf thymus RNA polymerase II is composed primarily of species IIA and IIB. These enzymes differ in the apparent molecular weight of their largest subunit, designated IIa and IIb for enzyme forms IIA and IIB, respectively. Both enzyme forms contain an additional high molecular weight subunit designated IIc. The structural relationship between subunits IIa, IIb, and IIc, labeled with 125I under both native and denaturing conditions, has been analyzed by two-dimensional peptide mapping. Native RNA polymerase II was iodinated and subunits IIa, IIb, and IIc purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The subunits were then digested with either trypsin or thermolysin and the 125I-labeled peptides resolved by thin layer electrophoresis in the first dimension and chromatography in the second dimension. Similar peptide maps were obtained for each of the three large subunits, suggesting that subunits IIa, IIb, and IIc are related in primary sequence. Alternatively, RNA polymerase subunits IIa, IIb, and IIc were purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, eluted from the gel, and then iodinated. The use of denatured subunits as substrate for the iodination eliminates the differential reactivity of specific tyrosine residues imposed by the structure of the native protein. Under these labeling conditions, the tryptic and thermolytic peptide maps of subunits IIa and IIb are nearly identical but bear much less resemblance to the peptide maps of subunit IIc than with the previous labeling procedure. These results suggest that subunits IIa and IIb are closely related in primary sequence but cannot establish whether these subunits are the products of closely related genes or are related by processing at the level of primary transcript or primary translation product. Subunit IIc bears a more distant relationship to subunits IIa and IIb. Possible reasons why this homology is only apparent in peptide maps from subunits labeled in the native enzyme are discussed.