To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified V J rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive V J rearrangements were sequenced. Nearly every functional V gene segment was used in rearrangements, although six V gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially. Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms. Utilization of J segments was also nonrandom, with J 1 and J 2 being overrepresented and J 3 and J 5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two V J rearrangements, marked differences were noted in the V segments used for the initial and subsequent rearrangements, whereas J segments were used comparably. Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the V J rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements. More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in V sequences derived from CD5Ϫ as compared with CD5