Erythroid precursors were obtained from the spleen of mice made anemic with phenylhydrazine and used to study the binding of bioactive 125I-rEp to membrane receptors. Kinetic analysis, using splenic cells obtained at different times following the induction of anemia, showed that the maximum binding was reached at day 3, and decreased thereafter; minimal amounts of 125I-rEp were bound to the splenic cells of normal mice. Splenic cells of day 3 anemic mice were fractionated using continuous Percoll density gradients, resulting in a fraction enriched in CFU-E (delta = 1.065-1072 g/ml) which showed the highest 125I-Ep binding on a per cell basis. The amount of 125I-rEp bound was greatly reduced in the densest fractions, which were comprised of maturing erythroblasts. The binding was time- and temperature-dependent, and a significant correlation was found with cell concentration up to 12 X 10(6). The amount of radioactivity specifically bound rose with increasing concentrations of 125I-rEp until a plateau was reached (2.5 nM), whereas non specific binding increased slightly and linearly. The binding of 125I-rEp was susceptible to competitive inhibition by unlabeled rEp, while other hematopoietic growth factors were ineffective. The calculated receptor density on these purified immature erythroid progenitors was 570 molecules with a Kd = 0.5 nM. Overall, these results suggest that the expression of the Ep receptor is reduced with increasing maturation.