Most studies of mammalian reticulocyte maturation have used blood reticulocytes. Nascent reticulocytes, as found in bone marrow, have not been available in developmentally synchronized populations. Nascent murine reticulocytes formed in vitro by enucleation of Friend virus-infected erythroblasts were purified and recultured for 110 hours. At 0 hours, all recultured cells were lobulated and contained dense, centralized reticulin. By 110 hours, about 20% to 25% of the cells became biconcave erythrocytes. Most ribosomes and cellular RNAs were degraded within 20 hours, and during that period, heme synthesis declined from a rate equal to that of late erythroblasts to less than 10% of that rate. Many mitochondria appeared normal until they showed outer membrane swelling, degradation, and apparent fusion with intracellular vacuoles at 40 hours of culture. During the period of mitochondrial loss, Bcl-X(L), an antiapoptotic protein that accumulates during erythroblast differentiation and maintains mitochondrial membrane integrity, demonstrated progressive decreases and changes consistent with deamidation. Nevertheless, the reticulocytes did not undergo apoptosis, because their apoptotic machinery was degraded. This experimental system that provides a developmentally synchronized population of nascent murine reticulocytes that mature into biconcave erythrocytes in vitro should be useful in further investigations of the cellular events involved in reticulocyte maturation.