Ig repertoires generated at various developmental stages differ markedly in diversity. It is well documented that Ig H chain genes in human fetal liver are limited with regard to N-regional diversity and use of diversity elements. It is unclear whether these characteristics persist in pre-B cell H chain genes of adult bone marrow. Using Ig H chain CDR3 fingerprinting and sequence analysis, we analyzed the diversity of Ig H chain third complementarity determining regions (HCDR3) in adult bone marrow pre-B and mature B lymphocytes. Pre-B cell HCDR3 sequences exhibited adult characteristics with respect to HCDR3 size, distribution of N regions and usage of diversity elements. This suggested that pre-B cells in adults are distinct from fetal B cell precursors with regard to Ig H chain diversification mechanisms. At the DNA sequence level, HCDR3 diversity in mature B cells was similar to that in pre-B cells. Pre-B HCDR3s, however, frequently contained a consecutive stretch of hydrophobic amino acids, which were rare in mature B cells. We propose that highly hydrophobic pre-B HCDR3s may be negatively selected on the basis of structural limitations imposed by the antigen binding site. At the same time, usage of hydrophilic HCDR3 sequences (thought to support HCDR3 loop formation) may be promoted by positive selection.