We have examined the appearance of thymocytes expressing gamma delta TCR within the developing thymus by using immunohistochemical techniques and flow cytometry in conjunction with the mAb 3A10, which recognizes a determinant associated with the constant region of the delta-chain. gamma delta+ Cells were first detected at day 16 of gestation, attained maximal levels at day 17 of gestation, and declined thereafter. By using the Ulex europeus agglutinin to identify medullary epithelial cells in situ, we observed a striking colocalization of gamma delta+ thymocytes and U. europeus agglutinin-positive medullary epithelial cells during late fetal and neonatal periods of development. In the thymuses of adult mice, gamma delta+ thymocytes were scattered throughout cortical and medullary areas of the thymus and most concentrated in the subcapsular areas of the thymus. Ultrastructural immunohistochemistry confirmed the close association between medullary thymic epithelial cells and gamma delta+ thymocytes in the neonatal thymus and also showed that some TCR-gamma delta molecules were patched to areas of contact with medullary epithelial cells. In contrast to the cellular distribution of either CD3 molecules or the TCR-alpha beta, where extensive intracellular labeling of thymocytes has been observed, cytoplasmic accumulation of delta-chain was not detected.