Treatment of pregnant C57Bl/6 mice with 48 mu g/kg diethylstilbestrol (DES) on gestation days (GDs) 14 and 16 resulted in both decreased day 18 fetal thymic cellularity as well as alterations in thymocyte phenotype. Histopathologic examination of GD 18 fetal thymi from DES-exposed dams demonstrated a decrease in thymic size and cellularity and an increase in pyknotic nuclei, indicative of apoptosis, relative to control thymi. Thymic architecture was also altered by DES treatment with a decrease in the distinction between the cortical and medullary regions. Flow cytometric staining of day 18 thymocyte suspensions with the apoptotic marker 7-aminoactinomycin D showed a decrease in thymocyte viability after DES, and a concomitant increase of thymocytes in early apoptosis. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, trends toward increased apoptosis were present in the CD4+CD8+ and CD4+CD8- subpopulations, and significantly increased apoptosis occurred in the CD4-CD8- and CD4-CD8+ subpopulations. These histopathologic and flow cytometric findings support enhanced apoptosis of thymocytes as a contributing factor to fetal thymic atrophy caused by DES.