Epidermal growth factor (EGF) receptors were studied during the in vitro differentiation of human trophoblast cells from first- and third-trimester placentas. Cytotrophoblasts were isolated by enzymatic digestion and purified on a discontinuous Percoll gradient. As analyzed by flow cytometry, 5 % of the cells are in the G<sub>2</sub>M phase in the early placenta and 0% in the term placenta. In culture, the cytotrophoblasts at both gestational ages flatten out, aggregate, and fuse together to form syncytiotrophoblasts. This in vitro morphological differentiation is associated with a threefold increase in the ability to bind specifically <sup>125</sup>I-EGF. Trophoblastic cells from the term placenta have a significantly (p < 0.005) higher receptor number (68.6 ± 9.5 fmol/mg protein) for EGF after 2 days of culture than first-trimester cytotrophoblasts (35.8 ± 2.3 fmol/mg protein). Scatchard plot analysis revealed two classes of binding sites with a similar affinity in both first-trimester and term placentas (9.5 × 10<sup>9</sup> M<sup>_1</sup> for the high-affinity, 0.5 × 10<sup>9</sup> M<sup>-1</sup>for the low affinity site). When <sup>125</sup>I-EGF was affinity cross-linked to cytotrophoblasts, the receptors appeared as a specific band with a molecular weight of 180 kD in SDS-PAGE. This study demonstrates that the culture of cytotrophoblasts offer an appropriate model to study the modulation of EGF receptors.