The purpose of this study was to define the zonal DNA content distribution in the basal versus the superficial crypt cells in Barrett's esophagus (BE) and related neoplastic lesions. One hundred and five tissue sections of BE patients and 12 gastric tissue section as controls were stained with hematoxylin-eosin and Feulgen and high-fidelity DNA histograms were generated from whole crypts (n=117) and also separately from the basal and superficial portions of the crypts (n=71). Three parameters were analyzed: (1) peak DNA index (DI), classified into diploidy (DI=0.9-1.1) or aneuploidy (DI>1.1), the latter of which was further divided into 3 types: mild (DI=1.1-1.3), moderate (DI=1.3-1.8), and severe (DI>1.8). (2) Heterogeneity index (HI), representing groups of cells with different DNA content. (3) Percentage of cells with DI exceeding 5N rate (5N-ER). In full crypts, compared with gastric controls, the prevalence of DNA aneuploidy increased significantly (P<0.01) from nondysplastic BE to basal crypt dysplasia (BCD), low-grade dysplasia (LGD), high-grade dysplasia (HGD), and adenocarcinoma (AC). Nondysplastic BE, BCD, and LGD had mostly mild aneuploidy, and the majority of HGD and AC had either moderate or severe aneuploidy. In addition, both HI and 5N-ER increased progressively from BCD and LGD to HGD and AC (P<0.01). When analyzed separately, the superficial crypt cells were diploid in nondysplastic BE and BCD, but were aneuploid in 50% of LGD and 100% of HGD cases. In contrast, basal crypt cells were aneuploid in 37% of nondysplastic BE, 50% of BCD, 73% of LGD, and 100% of HGD cases. A similar progressive increase in the HI and 5N-ER values in basal crypt cells was observed with dysplastic progression. The changes in DNA ploidy profiles of basal crypt cells in BCD and LGD were remarkably similar. These results suggest that with neoplastic progression, dysplastic changes in BE begin in the basal crypt cells and then extend further up the crypts, and BCD represents a true early form of dysplasia limited to the crypt bases.