AbstractPrevious studies from this laboratory have revealed an antigenic site located on the variable domain of the λ2 light chain of BALB/c myeloma protein 315 (the Vλ2315site). This site is recognized by conventional carrier‐specific T helper cells (Th) of BALB/c mice and is expressed on both the free and assembled Vλ2315domain. The present work defines two new antigenic sites associated with murine λ chains. The first site was associated with free λ1‐chain of myeloma protein J558. It was recognized by splenic Thfrom animals that had been primed with free λ1J558in complete Freund's adjuvant; when transferred to irradiated animals the primed Thresponded to a boost with (4‐hydroxy‐5‐iodo‐3‐nitro‐phenyl)acetate (NIP)‐free λ1J558in saline, but did not respond to NIP‐complete J558 or NIP‐free λ2315. Priming with complete J558 failed to elicit Ththat responded to NIP‐free λ1J558. This determinant was therefore only expressed on the free (as opposed to the assembled) form of λ1J558, and it was not shared with free λ2315. The second antigenic site was shared between free λ1J558and free λ2315. It was defined by free λ2315‐primed Thwhich responded to a boost with NIP‐free λ1J558. Since priming with free λ1J558did not elicit Ththat recognized NIP‐free λ2315, the cross‐reaction was undirectional. The free λ2315‐primed Thfailed to respond to the complete J558, and M315‐primed Thfailed to respond to NIP‐free λ1J558, indicating that the second (cross‐reactive) antigenic site, like the first, was only expressed on free λ chains. Completely reduced and alkylated (unfolded) free λ1J558and free λ2315chains elicited Ththat recognized native (folded) free chains.Thus, free λ1J558bears two antigenic determinants recognized by Th, one private and a second shared with free λ2315. λ2315also bears two determinants, a cross‐reactive one on free λ2315shared with free λ1J558, and a private one located on the Vλ2315domain of the complete M315.The discussion is focused on possible explanations for the quenching of the two new λ chain determinants upon light‐heavy chain assembly and why