Direct evidence was obtained for the existence of a specific high affinity alpha-fetoprotein (AFP)-binding protein in the cytosol of both MCF-7 human breast cancer cultured cells and primary breast cancer tissue from postmenopausal women using a nitrocellulose blotting assay. Scatchard analysis of the binding data for MCF-7 cells at 37 degrees C revealed the presence of a single class of AFP binding sites with an apparent Kd of 4.5 x 10(-8) M, and 75,000 binding sites per cell. All 9 primary breast cancer cytosols obtained from postmenopausal women also contained measureable levels of this specific AFP-binding protein. The number of AFP molecules specifically bound varied considerably between patients and ranged from 29-250 fmol per mg cytosol protein. Levels of AFP-binding protein levels and estrogen receptor measured in these same breast cancer cytosols showed a positive statistical correlation (r = 0.85). Taken together, the present evidence for the existence of a specific cytoplasmic AFP-binding protein in MCF-7 cells and previously reported evidence for de novo synthesis of free immunoreactive and bound nonimmunoreactive forms of cytoplasmic AFP by MCF-7 cells is consistent with the conclusion that most of the endogenous AFP synthesized in breast cancer cells is rapidly bound to specific cytoplasmic AFP-receptors, and that binding of AFP to these receptors masks its immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)