Estradiol receptor (ER) and progesterone receptor (PgR) were assayed in tumors from 20 patients with primary colorectal cancer. Ten of 20 tumors contained high affinity sites for 17 beta-estradiol and progesterone. The highest concentration of ER was 56 fmol/mg of protein. The ER dissociation constant ranged from 1.6 X 10(-10) M (mean 4.6 +/- 2.6). The highest concentration of PgR was 42 fmol/mg of protein. The PgR dissociation constant ranged from 3 X 10(-9) to 9 X 10(-9) M (mean 5.65 +/- 2.1). Four out of 20 specimens analyzed were from male patients and all resulted negative for both receptors. Sixty per cent of ER positive tumors were also PgR positive, whereas only 20% of ER negative were PgR positive. Sucrose gradient centrifugation showed that cytoplasmic ER of colorectal cancer sedimented at 3 S in the absence of protease inhibitors and at 4.5 S in the presence of 1 mM phenylmethylsulphonyl fluoride (PMSF) both in low and in high ionic strength. When chromatographed on Sephadex G-200 almost all ER was quantitatively recovered in the included fractions. Molecular weights of ER eluted from Sephadex G-200 ranged from 90,000 to 50,000 daltons. Elution profile and molecular weight heterogeneity suggest that, in spite of the presence of PMSF, there is a limited proteolysis of ER. Partially purified colorectal cancer ER did not bind to sepharose-heparin. The isoelectric point of ER was 6.4-6.5.