Expression of the surrogate light (ψL) chain genes encoding the VpreB and λ5/14.1 proteins is restricted to B-lineage cells. Pro-B and pre-B cells produce ψL chains, but whether both employ these as cell surface receptor components remains enigmatic. Recombinant human VpreB protein was used to generate a large panel of monoclonal anti-VpreB Abs to examine this issue. Native ψL chain proteins within pro-B cells as well as those serving as receptor components on pre-B cells were precipitated by 16 of the 26 anti- VpreB Abs. Surrogate light chains were easily detected on pre-B cell lines, whereas these anti-VpreB Abs reacted with pro-B cell lines only after plasma membrane permeabilization. The subpopulation of normal bone marrow cells bearing pre-B receptors included large and small pre-B cells exclusively, although pro-B cells also contained intracellular VpreB. VpreB proteins were not detected on or within B cells in bone marrow or the circulation, but a subpopulation of B cells in germinal centers was found to express the VpreB proteins intracellularly. Surrogate L chains are thus intermittently produced during human B-lineage differentiation, while their role as receptor components appears limited to the pre-B cell stage.