Although the amino acid sequence and three-dimensional structure of human immunoglobulin light chains have been known for more than 15 years, the location of antigenic markers characteristic of lambda chains has not been determined. Here, we use a set of synthetic overlapping peptides to completely model the sequence of the lambda chain Mcg and test these for the binding of rabbit and goat antisera specific for lambda chain determinants. We assess peptide contributions to lambda-antigenic reactivity and also to identify a portion of C-region where conformational factors contribute to the antigenicity. Specific determinants occur both in the constant and variable (first and third framework) domains of the molecule. The fourth framework of the variable region, a segment specified by the joining gene, is also recognized and cross-reacts antigenically with the homologous region of T cell receptor beta chains. Major lambda specific determinants are localized in the N- and C-terminal segments, which are linear and devoid of major conformational folding. Other segments that are strongly antigenic, such as the third framework of the V region (residue 78-93) and a segment of the constant region (residues 177-192), show strong conformational dependence in antigenicity.