To determine the Vkappa gene utilization in cord blood, we made libraries of Igkappa sequences from two cord blood cDNA samples. The rearranged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all Vkappa genes. Although the human kappa locus contains approximately 38 potentially functional V genes, we observed that approximately 75% of the 146 sequences from our two samples used only nine Vkappa genes. Using leader-specific primers, we also amplified VkappaI and VkappaIII rearrangements from genomic DNA from one of these individuals. Nonproductive rearrangements give an approximation of the relative frequency of gene rearrangement. Some of the genes that were overused in the cDNA libraries were also observed to rearrange frequently, but others did not show high rearrangement frequencies, suggesting that cellular selection caused their increase in the periphery. Surprisingly, we observed a high frequency of rearrangements using L9, which has been reported to be a defective Vkappa gene. Sequence analysis of the unrearranged gene revealed two new functional alleles of this gene. We observed that N nucleotides were present in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J junctions of rearrangements of two nonfunctional V genes. Forty-six percent of those cord blood sequences contained N regions. In comparison, 57% of junctions of the rearranged nonfunctional gene from adult PBMC contained N regions. Finally, we observed that CDR3 length heterogeneity was more pronounced for VkappaIII genes than for all of the other Vkappa families.