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Epithelia and fibromuscular walls were dissociated from the vaginae of ovariectomized BALB/cCrgl mice (ca. 41 days old) exposed neonatally to diethylstilbestrol (DES) or sesame oil and purified by centrifugation through Percoll density gradients. Neonatal exposure to DES caused the vaginal epithelium to become permanently proliferated and partially keratinized; the control epithelium was low cuboidal. The major cellular proteins expressed in each tissue compartment were examined by two-dimensional gel electrophoresis of [35S]methionine-labeled tissues. The epithelia and fibromuscular walls displayed distinctive two-dimensional protein patterns. In the DES-exposed vaginal epithelium, the expression of two proteins (one had a molecular size of 65 kDa and a pl of 6.0, and the other had a molecular size of 38 kDa and a pl of 6.3) was increased, while the expression of three proteins with molecular sizes of 25, 30, and 140 kDa and pls of 5.6, 5.6 and 6.7, respectively, was reduced, relative to the control epithelium. In the DES-exposed vaginal fibromuscular walls, the expression of 9 proteins was increased whereas the levels of 21 specific proteins, distinct from those in the epithelium, were decreased. Thus, long-term tissue-specific alterations in the synthesis of a select number of cellular proteins occur in the DES-exposed vagina.