Proliferating cells require iron and, therefore, express the transferrin receptor (CD71) that mediates cellular iron uptake. Cycling thymocytes, which have the CD4-8-3-, CD4-8+3-, or CD4+8+3- phenotypes, also express CD71. The importance of CD71-mediated iron uptake for proliferation and maturation of thymocytes was studied using fetal thymus organ cultures at day 14 of gestation and treating them for 7 days with a CD71 monoclonal antibody (mAb). The intracellular iron deficiency caused by this treatment, inhibits both proliferation and maturation of the thymocytes. Cell recovery was reduced by 60%, but cells still expanded tenfold during the culture. Remarkably, the final maturation of alpha beta T cells was completely blocked as no thymocytes with low or high CD3/alpha beta TcR expression developed. Moreover, only few cells reached the CD4+8+3- stage of T cell development. CD4-8-3- thymocytes, however, as well as its CD44-25+ subset developed in normal numbers, suggesting that CD44-25+ CD4-8-3- cells, or their immediate progeny, were most vulnerable to CD71 mAb treatment. The development of gamma delta T cells, which also express CD71, was not affected in these cultures. This suggests that gamma delta T cells are either less iron-dependent or possess alternative iron-uptake mechanisms. Thus, our observations indicate that CD71 treatment, causing decreased intracellular iron levels, severely inhibits the major proliferation phase from the CD44-25+ CD4-8-3- to the CD4+8+3- cells, and completely abrogates the final maturation of CD4+8+3- cells into alpha beta TcR-expressing cells. In contrast, proliferation and differentiation of the earliest thymic precursors into CD44-25+ CD4-8-3- cells is not affected by CD71 treatment.