Primary breast adenocarcinomas obtained from ten patients were enzymatically digested using collagenase (1 mg/ml), hyaluronidase (1 mg/ml), elastase (0.1 mg/ml) and DNAse (0.2 mg/ml). The tumor cells were labeled with 3H-thymidine and, in some cases, with 3H-estradiol. The isolated cells were submitted successively to a Ficoll-Hypaque and a bovine serum albumin gradient, from which 12 fractions were obtained. In each fraction, several characteristics were determined: carcinoembryonic antigen (CEA), thymidine (dThd) incorporation, and estrogen receptors (ER). Three main cellular subpopulations were characterized: An intermediate density subpopulation (1.046-1.054 g/ml), in which the proliferating cells are concentrated. In this subpopulation a small number of CEA-positive cells are present, but ER containing cells are virtually absent. A high-density, small cell subpopulation that concentrates most of the ER-containing cells. This subpopulation lacks proliferating cells, but CEA-containing cells are abundant. A low-density subpopulation, lacking proliferating cells and with scarce ER-positive cells, although CEA-positive cells are frequent. These findings strongly suggest that proliferating cells lack ER.