Mouse spleen lymphoblasts, stimulated to divide in vitro, acquired a low cell density and could be separated by isopycnic techniques. Cultured cells were suspended in BPA columns, rho = 1.080, and spun to equilibrium. The method was simple, fast, accomodated large numbers of cells, and was reproducible. It provided lymphoblasts in high yield and purity (at least 80% of the low density cells were blasts). It allowed for the recovery of proliferating cells in their first cell cycle, and did not alter the subsequent ability of cells to proliferate when recultured in vitro. Certain properties of mouse spleen lymphoblasts were analyzed in detail. Lymphoblasts induced by LPS, FCS, con A (tetravalent and succinylated), and MLC were very similar except in the absolute numbers that were induced. The blasts exhibited the classic cytologic features of enlarged nucleoli and abundant cytoplasmic polyribosomes (basophilia). As a population, they were enlarged in size relative to nondividing cells, but this seemed to apply primarily to cells in the S and G2+ M phase of the cell cycle rather than G1. The cell cycle distribution of lymphoblasts was analyzed by flow microfluorometry. By analyzing low density cells obtained at varying intervals after mitogen stimulation, FMF indicated that lymphoblasts enter the S phase of their first cell cycle beginning at 20-24 h after stimulation.