Mild reduction of mature, thymic Ly-2/3 heterodimers of Mr 67 000 resulted in dissociation into three individual polypeptide chains, alpha, alpha', and beta, of respective Mr values 38 000, 35 000, and 30 000. The alpha and alpha' chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the beta chain. The possibility that the alpha and beta chains of each heterodimer established their interchain links within a labile precursor protein in which alpha and beta segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment - the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.