In the present study, the effects of cell cycle phase and proliferation rate on the expression of specific estrogen binding activity were explored in hormone-dependent human breast cancer cells. A technique was developed to alter the proliferative rate of MCF-7 cells by plating at different densities. The doubling time ranged from 20 to 48 hr, showing a negative relation to the number of plated cells. Slowly proliferating cells had accumulated more than twice as much estrogen receptor (ER) activity as did fast-proliferating cells. Exposure of exponentially growing cells to isoleucine-deficient medium resulted in decreased thymidine incorporation and disappearance of detectable cellular ER activity. Overall protein synthesis was reduced by only 30% in cells growing in isoleucine-free medium. At 24 hr after release from isoleucine deprivation, ER levels are fully restored, although thymidine incorporation does not resume for an additional 6 to 8 hr, and increases in cell number are not seen for 24 hr. Exposure of exponentially growing cells to 2 mM thymidine for 24 hr produced partially synchronized MCF-7 cells (approximately 70%). Six hr after release from excess thymidine, cells reached S phase; after 9 hr, G2; and after 18 hr, G1. ER levels immediately and, 6 hr after release, remained unchanged, showed a slight increase at 9 hr, and showed an increase of about 50 to 60% at 18 hr. These data suggest that: (a) ER binding activity and DNA synthesis can be dissociated; (b) ongoing protein synthesis is necessary for maintenance of cellular ER activity; and (c) ER is apparently synthesized throughout the cell cycle, with some evidence that this is predominantly in G1 and G2.