In this study we report on the characterization of a panel of 62 hybridomas generated by fusing unstimulated spleen cells from neonatal (less than 24 hr old) normal BALB/c mice with the non-secreting Sp 2/0 cell line. The vast majority (98%) of these hybridomas secreted Ig but only 20% produced IgM. The isotype of the remaining hybridomas was determined as being IgG2b. Interestingly, when splenocytes from 1-day-old mice were stimulated with LPS for 48 h prior to the fusion event, 84% of the hybridomas were secreting IgM. The hybridoma supernatants were screened either by ELISA or RIA for binding reactivity using a panel of 17 Ag, proportionally divided between self and non-self. A binding reactivity could be assigned in 44% of cases. Of these, 29% were monoreactive, i.e., reactivity occurred with one Ag only, while the remaining 15% were multireactive. The majority (21 of 27) of hybridomas with a defined reactivity were directed against self-Ag. These included autologous red blood cells, DNA, histone H1, thyroglobulin, and Ag of the cell surface of T cells. The frequency of utilization of VH genes was determined using DNA probes for eight VH gene families. While all VH gene families appeared to have been used, one, VH 7183, had a slight but significant (p less than 0.02) higher utilization than expected by random expression. The frequency of all the other VH gene families was not significantly different from random utilization. No correlation was found between Ag reactivity in the supernatants and the utilization of a particular VH gene family. These findings indicate that early in the ontogeny the predominant reactivity of B cells is for self-Ag and, unlike what it is commonly believed, the IgM isotype is not dominant within these endogenously activated B cells at this time of ontogeny when genes from all VH families are utilized.