Thymic precursor cells (prothymocytes) comprise a large proportion of the fetal thymic cell population, but are less frequently encountered in the postnatal thymus, where they compose < 1% of the entire population. In the present study we attempted to characterize a number of properties of the prothymocytes obtained from human fetal thymic tissues after depletion of the E-rosette thymocyes on a Ficoll-Hypaque gradient. The prothymocytes are larger than the thymocytes and show a different nuclear chromatin pattern. This subset of cells lacks the E-rosetting and natural-attachment capacities and, unlike thymocytes, does not bind the lectin peanut agglutinin. Human prothymocytes are highly sensitive to the in vitro cytolytic effect of hydrocortisone, whereas the thymocytes are resistant. Long-term in vitro culture of prothymocytes resulted in the expression of thymocyte characteristics together with a burst of mitotic activity. Results of this study indicate that the rate of the prothymocyte proliferation is regulated by the small thymocytes present in the same suspension.