On day 0, 30 isolated LSKCD34⁻ Ly5.1⁺cells were co-injected, together with 200 000 unfractionated Ly5.2⁺ bone marrow cells, into each lethally irradiated C57Bl/6 recipient. From the same isolations, cells were set in Terasaki plates at 0.25 cell/well using serum-free medium supplemented with KFM3; 12 to 16 h after seeding, wells were screened for the presence of 1 cell, and only wells containing 1 cell were included in experiments. On day 10, 30 wells containing cells that had proliferated were pooled and injected into each recipient, together with 200 000 unfractionated Ly5.2⁺ bone marrow cells. Reconstitution in peripheral blood was evaluated after 4 months. Data are shown as means (±SEM). Secondary transplantations were in experiment 1, performed by pooling cells from all primary recipients and injecting cells corresponding to a half-femur into 5 secondary recipients. In experiment 2, BM cells from each primary recipient was injected into 2 secondary recipients (total, 10 secondary recipients/group). In experiment 1, lineage analysis of donor-derived Ly5.1⁺cells revealed that 3 mice that received uncultured cells displayed only lymphoid reconstitution. In experiment 2 of the serially transplanted mice, 3 animals transplanted with BM from recipients of uncultured cells had too low reconstitution to allow meaningful lineage analysis. In all other mice, donor-cell reconstitution contributed to multilineage reconstitution.