The relationship of B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) was explored in vitro by examining the effect of hydroxyurea (HU), an inhibitor of cellular DNA synthesis, on the generation of ISC from human peripheral blood mononuclear cells (PBM). HU completely inhibited the capacity of PBM to generate ISC in response to pokeweed mitogen (PWM) and other polyclonal B cell activators. Inhibition resulted from an effect on B cell proliferation, because HU also prevented the generation of ISC in cultures of purified B cells supplemented with either T cell supernatants or mitomycin C-treated T cells. Inhibiting B cell proliferation by treating them with mitomycin C before culture also abolished the generation of ISC. When ISC were enumerated after a 7-day incubation with PWM, the addition of HU as late as day 6 of culture was found to inhibit responsiveness markedly. This suggested that those cells that had acquired the capacity to secrete lg were actively dividing, and continued division was necessary for ongoing lg secretion. To examine this possibility, experiments were carried out in which responsiveness was assayed on a daily basis. ISC could be detected after a 3- or 4-day incubation and reached maximum at day 6 or 7. Addition of HU on days 3 to 7 caused a highly significant reduction in the number of ISC within 24 hr. ISC did not begin to show resistance to the effects of HU until later in culture. This observation supported the conclusions that ISC were a rapidly cycling cell population and that ongoing lg secretion, as well as expansion in the number of ISC, depended on continued proliferation of the ISC. To confirm directly that ISC were a cycling cell population, PBM were cultured with PWM for 6 days, fixed, stained for both cytoplasmic lg and DNA content, and analyzed on the fluorescence-activated cell sorter. This method made it possible to quantitate the DNA content of individual lg-synthesizing cells and thus to determine their position in the cell cycle. As many as 40% of cytoplasmic lg-positive cells were found to be in the S, G2, or M phases of the cell cycle. These data indicate that ISC generated in man after in vitro stimulation with a number of polyclonal activators are not stable terminally differentiated lg-secreting plasma cells but rather an actively cycling lg-secreting population. Furthermore, the results indicate that proliferation of the ISC themselves plays an important role in determining the magnitude of the resultant antibody response.