The diversity of the immunoglobulin heavy (H) and light (L) gene libraries used to construct a combinatorial library is an important parameter in determining the characteristics of antigen-specific Fab obtained from the library. To investigate the role of library diversity, we compared Fab specific for the autoantigen thyroid peroxidase (TPO) isolated from two different combinatorial libraries. Both libraries contained the same H chain genes. The original combinatorial library (H/R) utilized kappa chains generated using a single kappa variable region oligonucleotide primer. We constructed a second combinatorial library (H/D) containing kappa chains amplified with a diverse panel of variable region primers. From the the original H/R library, only two groups of TPO-specific Fab had been obtained, involving two H chain types (V1-3B and hvlL1) but only one kappa chain type (012). In contrast, among the seven TPO Fab characterized from the second library (H/D) we observed five different VH/VL combinations, comprising three types of H chains (V1-3B, VH26 and DP7) and four types of kappa chains (O12, L12, L2/hv328H5 and B3). Besides differences in VH and VL genes, as well as VH/VL combinations, the new TPO Fab used different D regions and JH and JK elements. Nevertheless, the new kappa Fab resembled previously isolated TPO Fab in terms of their affinity for TPO (Kd~10−9M) and preferential recognition of conformationally intact autoantigen. In summary, our studies demonstrate that the diversity of the L chain library repertoire, while having little effect on immunological properties, has a major influence on the genes encoding antigen-specific Fab selected from a combinatorial library. For the successful isolation of rare but clinically important autoantibodies (such as to the TSH receptor) by the combinatorial library approach, library diversity is likely to be a major factor.