Only two sets of antigenic determinants are recognized by T lymphocytes at uniquely high precursor frequencies: those encoded by the MHC and those encoded by Mls. The structural as well as functional characteristics of MHC products have been extensively analyzed. In contrast, little information concerning the nature of Mls genes or their products is available. Although it was originally described (5, 6) that the Mls locus on chromosome 1 is composed of four alleles that encode polymorphic cell surface structures, the issues of polymorphism and allelism in the Mls system have been controversial for some time. In the present study, T cell clones were generated by continuous stimulation of B10.BR (H-2k, Mlsb) T cells by CBA/J (H-2k, Mlsd) stimulators and they were used to analyze the relationship of putative Mlsa, Mlsc, and Mlsd determinants. All clones proliferated in response to determinants expressed by CBA/J stimulators. In addition, each of these clones exhibited a second reactivity to either AKR/J (H-2k, Mlsa) or C3H/HeJ (H-2k, Mlsc) stimulators. No clone responded to both AKR/J and C3H/HeJ. These second specificities were defined to be for Mlsa or Mlsc determinants, respectively, by the response patterns of clones and unprimed T cells to stimulators derived from congenic strains, recombinant inbred (RI) strains, and backcross mice. Moreover, a segregation analysis of the (CBA/J X B10.BR)F1 X B10.BR backcross indicated that the Mlsa-like and Mlsc-like determinants expressed on CBA/J (Mlsd) cells are in fact encoded by nonallelic, unlinked genes. These findings suggest a new concept of the polymorphism and genetics of the Mls system. It is proposed that two distinct and nonallelic gene products express, respectively, the noncrossreacting Mlsa and Mlsc determinants, and that the Mlsd phenotype does not represent an independent genotype but rather reflects the concurrent expression of Mlsa and Mlsc. The Mls system, therefore, consists of at least two systems that are distinct both genetically and antigenically, and that may be of different biologic or physiologic significance as well.