The primary structures of the allelic products of H-2L, the murine "third locus" major histocompatibility antigen, were compared with the structures of the H-2K and H-2D antigens by tryptic-peptide analysis. The H-2Ld,H-2Lq, H-2Dd, H-2Dq and H-2Kd molecules were radiolabeled and isolated from spleen cell detergent lysates with specific alloantisera. Comparisons of the tryptic maps of the H-2L, H-2D, and H-2K molecules of the H-2d haplotype demonstrated that H-2Ld molecules were as different from H-2Dd molecules as H-2Dd molecules were from H-2Kd molecules. In contrast, the H-2Lq and H-2Dq molecules were found to be extremely similar and a second peptide separation method was required to discern structural differences between the 2 molecules. Comparisons of the allelic products H-2Ld and H-2Lq indicated possible structural differences despite the lack of a known serologic difference. These results, while raising questions about the genetic evolution of the H-2L locus relative to the H-2K and H-2D loci, demonstrate that the structure of the H-2L molecule is distinct from that of the H-2K and H-2D molecules. These structural analyses further substantiate the functional studies that define the H-2L molecule as the "third" major histocompatibility molecule of the mouse.