The experiments reported here were designed to answer two questions: (1) At what stage in normal pre-B cell development do immunoglobulin gene rearrangements occur?; and (2) Do heavy chain and kappa light chain genes rearrange in concert, or in an ordered sequence? To answer these questions, we studied immunoglobulin gene rearrangements in pre-B cell populations purified on the fluorescence-activated cell sorter (FACS). Gene rearrangement was assessed by measuring the loss of germ-line joining (J) segment-containing restriction fragments in B cells and two populations of pre-B cells. Large pre-B cells, the earliest identifiable cells in the B lineage, have rearrangements at both JH loci but do not have rearrangements at the kappa chain loci. Thus heavy chain rearrangement occurs concurrently with or prior to the expression of the surface marker B220, which we use to identify and isolate pre-B cells. Small pre-B cells, which include the immediate precursors of B cells, likewise have rearrangements at both JH loci, but may also have J kappa rearrangements. Approximately 1/3 of the J kappa loci are rearranged in small pre-B cells compared to 2/3 in kappa chain-expressing B cells. This suggests that the small pre-B cell population is actively undergoing kappa chain gene rearrangement. The striking asynchrony in heavy and light chain gene rearrangement is reflected at the level of gene expression; both pre-B cell populations synthesize mu chains but not kappa light chains. Heavy chain rearrangement is therefore a very early event in B lineage development and may begin in a cell not yet fully committed to the B lineage, whereas kappa rearrangement occurs just prior to the expression of surface immunoglobulin (sIg) and may be a rate-limiting step in the transition from pre-B to B cells.