Radioactive Ia.4 molecules were prepared from 3H- or 14C-labeled splenocytes, selected PEL, or bronchoalveolar macrophages (M phi). Studies in the accompanying paper indicated that incorporation into Ia.4 in these 3 populations is due to B cells, T cells, and macrophages, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the 58,000 m.w. form of Ia.4. The eluted molecule was then reduced, and the resultant alpha and beta subunits were isolated by a second separation on SDS-PAGE. Alpha (or beta) chains from 1 population labeled with 3H-amino acids was mixed with alpha (or beta) chains obtained from a population representing a different cell type that was labeled with 14C-amino acids and the mixture was digested with trypsin. Double-label (3H/14C) comparative peptide mapping was performed using high-pressure liquid chromatography to separate the peptides. Eighteen to 20 peaks of radioactivity were resolved from alpha chains, and 12 to 15 from beta chains. No reproducible differences were observed when comparing alpha or beta chains of T cells and macrophages, or those of B cells and macrophages. These results indicate that the primary structure of Ia.4 molecules is identical on the 3 cell types in question. The implications of having a T cell bearing the same Ia that it recognized on a macrophage in conjunction with antigen is discussed.