Significantly less long-term culture initiating cells (LTC-IC) are recovered from cultures in which progenitors are cultured in contact with stroma (stroma-contact) than when cultured separated from stroma by a transwell (stroma-noncontact). This suggests that direct contact with stroma inhibits either proliferation or survival of LTC-IC. Using the membrane intercallating fluorochrome, PKH-26, we demonstrated that significantly less LTC-IC plated for 14 days in stroma-contact cultures proliferated than in stroma-noncontact cultures (16+/-7 vs 50+/-10%). Furthermore, when LTC-IC were sorted singly in stroma-contact cultures for 2 weeks, only 25+/-4% of individual LTC-IC progeny could initiate two secondary stromal cultures and had therefore proliferated, whereas 45+/-6% of single sorted LTC-IC progeny proliferated when cultured in stroma-conditioned medium without stromal feeder. However, LTC-IC survival was similar in both culture systems. Finally, proliferation inhibition occurred even when LTC-IC were cultured in contact with glutaraldehyde-fixed stroma, which is no longer capable of producing growth inhibitory or stimulatory cytokines. Thus, direct adhesive interactions between LTC-IC and stromal components inhibits their proliferation.