Populations of cells with different biochemical characteristics can be obtained following enzymatic digestion of newborn mouse calvaria. Previous studies had suggested an enrichment for periosteal fibroblasts in early released cells, based on their proliferative response to PGE2 and for osteoblasts in late released cells due to their high cAMP response to PTH. Intermediate cells were presumed to be a mixture of those two phenotypes. We have continued our characterization of these populations by studying their mitogenic responses to IGF-1 and EGF since these polypeptide growth factors have been reported to stimulate proliferation of immature bone cells in organ cultures of bone. [3H]Thymidine incorporation into acid insoluble radioactivity was stimulated by IGF-1 in all populations but the greatest increase was in intermediate populations. These latter cells were also the most highly responsive to EGF, which had a small growth stimulating effect on early released cells and no significant effect on late released cells. In bone cells both low and high affinity EGF binding sites were identified (Kd approximately 4 nM and 0.5 nM) and the total EGF binding sites/cell were approximately two-fold higher in intermediate than in early and late released populations. These data suggest that among sequentially released populations of bone cells the intermediate populations may be enriched for immature bone cells from the proliferative zone of bone.