In order to study the fine structure of H-2L, the third H-2 locus of the mouse major histocompatibility complex, we have produced and characterized the monoclonal anti-Ld antibodies secreted by 3 different hybridoma cell lines. Two antibodies were obtained by a specific immunization directed to Ld determinants. Another antibody obtained from an immunization including, but not specific for, an L locus difference was found to detect Ld, and not Kd or Dd molecules. Although the fusions involved an H-2d myeloma variant line, no apparent adverse "autoreaction" was observed in these anti-H-2d antibody-producing hybridoma cells. All 3 antibodies exhibited cross-reactions with the antigens of certain haplotypes, and the reactivity patterns observed corresponded to the previously described alloantigenic specificities H-2.64 and H-2.65. This concordance with known H-2 specificities contrasts with the broad, unique specificity patterns exhibited by most monoclonal antibodies to H-2 antigens and may reflect a difference in the complexity of the polymorphism of the L locus relative to that of the K and D loci. Immunoprecipitation analyses using 1 of the monoclonal antibodies showed that the molecules precipitable by this reagent were indistinguishable from those precipitable by anti-Ld alloantisera. In addition, cross-reactions of Ld, Dq, and Lq molecules were revealed by sequential immunoprecipitation analyses, supporting a close evolutionary relationship of L and D loci.