The effects of erythropoietin (epo) on the proliferation of late erythroid progenitor cells (CFU-E) and on the formation of hemoglobin and of globin mRNA in these cells are described. CFU-E were isolated from thiamphenicol-pretreated anemic mice by elutriation and Percoll density gradient methods. These CFU-E are restricted in their capacity to proliferate in vitro without added epo. The epo dependence in vitro was not absolute. With no epo in the culture medium the first cell division was unimpaired, whereas the third division was only 1%-2% of the control. In the absence of epo the synthesis of hemoglobin is very low in CFU-E, but is increased significantly after about 5 h of incubation with epo present. In epo deprived cells there was considerable hemoglobin formed at about 14 h, but not earlier. The presence as detected by the Northern blot technique of globin mRNA, isolated from CFU-E, was variable, probably depending on the presence of some more mature erythroid cells. By an extrapolation method we show evidence that pure CFU-E would have virtually no detectable globin mRNA. The production of globin mRNA is rapidly (2 h) induced in cells incubated with epo. We conclude that epo, besides having a mitogenic effect on CFU-E, induces the rapid expression of the globin genes.