Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area, CA) beneath the stromal layer was weekly determined for at least 8 weeks, and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size, but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples, ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM, 8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU, and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells, whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion, the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases.