Determining the mechanisms by which B cells develop that culminates in the generation of a diverse repertoire is critical to our understanding of how the immune response is regulated. The B cell specificity repertoire appears to be developmentally acquired in a predetermined, temporally ordered fashion. Thus, in the Balb/c mouse, the ontogenetic appearance of functional B cells specific for various hapten probes occurs in the order of dinitrophenol (DNP), fluorescein (FI), and phosphorylcholine (PC). In addition, when the influenza virus hemagglutin molecule was used as an antigen probe, similar conclusions were drawn regarding a patterned acquisition of the specificity repertoire. More recently, we have used a fetal organ culture system to show that hapten-responsive B cells appear in the same predictable, temporal order in vitro. The importance of these studies was that the effects of environmental influences were minimized in the absence of circulation and cell migration, and therefore the results indicated that the patterned emergence of the specificity repertoire observed was largely the result of genetic regulatory processes. Recent molecular findings may relate to such genetic regulatory processes. The V(H) genes in Balb/c mice have been grouped into eight families based on sequency homology, and have been mapped relative to the constant region genes. Yancopoulos et al. and Perlmutter et al. have shown that the V(H) gene segments closest to the J(H) cluster, the V(H1B) 7183 family, are preferentially utilized in transformed fetal pre-B cell lines and in fetal B cell hybridomas. This led to the hypothesis that the developmental control of the expression of V(H) gene segments is related to chromosomal organization. A logical extension of these findings is that the programmed appearance of particular clonotypes in ontogeny may be explained, in part, by the preferential use of particular V(H) gene segments. However, to what extent the transformed B cell lines represent members of the functional expressed repertoire could not be evaluated. In the studies described herein, the fetal organ culture system was used to assess the early expressed repertoire at the clonotypic level using idiotypic analysis. Anti-DNP secreting clones were derived from fetal organ cultures and tested for the presence of two idiotypes, 36 and MOPC 460 (460). The 36 idiotype is a predominant DNP clonotype of the neonatal repertoire, while the 460 idiotype is a major cross-reactive idiotype of the adult DNP response. We found that a large proportion of the DNP-specific fetal B cell clones express both the 36 and 460 idiotypes with as many as 40% of midgestation cultured DNP-specific fetal B cells expressing the 460 idiotype. Both of these idiotypes are associated with V(H) segments from the 36-60 family. Therefore, the results indicate that at least half of the early functional DNP-specific B cell repertoire uses the V(H) 36-60 gene family.