It has been previously demonstrated that normal nonimmunized individuals possess circulating and tissular B cells, which are capable of spontaneous immunoglobulin (Ig) production in short-term (3 days) cultures. We have also observed the occurrence of low levels of [3H]thymidine uptake early in such cultures. This work analyzes the relationship between both spontaneous B-cell functions in vitro: Kinetics studies revealed that both activities were temporarily related, as spontaneous DNA synthesis was maximal from 8 to 12 hr, and declined thereafter, when spontaneous Ig secretion was first detected in the culture supernatant: The abrogation of DNA synthesis at the culture initiation or during the period of early proliferation, but not after 24 hr, inhibited subsequent IgG secretion. The B cells responsible for spontaneous DNA synthesis and IgG secretion exhibited similar low densities, since both were recovered in the 42.5-45% Percoll fractions, and identical large size as determined by 1g sedimentation procedure, and in tonsil, were equally reactive with the BA-2 mouse monoclonal antibody. Finally, limiting dilution analysis showed that the precursor frequencies of both cells under study were similar. These results suggest that spontaneous DNA synthesis and IgG production are carried out by the same subset of in vivo-preactivated lymphoblastoid B cells.