The control of cell proliferation by estrogens was examined under the premises of the indirect-negative hypothesis, which states that estradiol cancels the proliferative inhibition exerted by a serum-borne protein on estrogen-target cells. Fractionation protocols resulted in the co-elution of the inhibitory activity with serum albumin. Removal of human albumin (HA) from charcoal-dextran stripped serum by hexyl-S agarose chromatography resulted in a preparation lacking inhibitory effect. HA inhibited the proliferation of human estrogen-target MCF-7 cells. Human non-estrogen-target MDA-MB231 cells were impervious to the effect of HA. MCF-7 cells were exposed to recombinant human albumin (rHA) and to its rDomain I and rDomains I + II. Inhibition of proliferation was maximal with rHA and with rDomains I + II; rDomain I was less inhibitory. The inhibitory effect of albumin was cell type and protein specific. Only estrogens cancelled the albumin inhibition; recombinant growth factors and other hormones were ineffective. These data suggest that: (a) albumin or a portion of it (most likely within Domains I and II) is the specific inhibitory signal for the proliferation of human estrogen-target, serum-sensitive cells; (b) estrogens specifically cancel this inhibition; (c) inhibitory signals prevail over putative growth factors; and (d) the default state in these cells is proliferation.