Proliferation of a human carcinoma cell line (RPMI-2650) was found to be density dependent in culture In a double-layer agar clonal assay, no growth was observed below a critical "cut-off" density unless autologous feeder cells were present. Medium conditioned by RPMI-2650 cells could substitute for the physical presence of feeder cells, thus opening the way to biochemical analysis of the nature of the feeder and density dependence effects RPMI-2650 conditioned medium (CM) was shown, using specific cell culture bioassays, to contain TGF-a (transforming growth factor-a)-like, TGF-B-like and autostimulatory activities, as well as EGF (Epidermal growth factor) receptor binding activity. Following large-scale production of CM m roller and suspension culture, these activities were fractionated using ultrafiltration, gel filtration m acetic acid and HPLC HPLC separated TGF-a and TGF-B activities, but autostimulatory activity was lost RPMI-2650 cells had lower EGF receptor levels than'3 other human carcinoma cell lines examined (A431, T-47D, SCC-9). EGF receptor levels m some of these lines were shown to be regulated by steroid hormones. The results presented here indicate that the autostimulatory activity may consist of at least two components, one of high molecular weight (> 30 kDa) and another of low molecular weight (< 1 kDa), and may be distinct from previously-described TGFs.