In 10 cases of Barrett adenocarcinoma, samples from 8 tumor areas (including superficial and deep from peripheral and central areas) and a regional lymph node metastasis were studied for amplification of c-myc, c-erbB-2, and EGFR. We analyzed loss of heterozygosity (LOH) at 3 loci (APC, MCC, and RB) and 2 anonymous microsatellite markers (D4S1652 and D18S474). We detected c-myc in variable fractions of tissue samples from 3 of 9 tumors; EGFR was amplified in 2 specimens from 1 tumor. One tumor demonstrated amplification of c-erbB-2 in all areas. LOH at the D4S1652, MCC, RB, APC, and D18S474 loci was found in 75% (3/4), 57% (4/7), 50% (4/8), 11% (1/9), and 0% (0/10) of informative cases, respectively. LOH generally was restricted to variable subpopulations of tumor cells within individual tumors. There was no obvious association of certain genetic alterations with topographically distinct tumor regions; however, superficial areas showed more frequent genetic alterations than areas from the deeply invading front. More aberrations were detected in the periphery than in the center. Barrett adenocarcinoma is characterized by marked intratumoral genetic heterogeneity, which must be considered when evaluating genetic alterations as indicators of response to therapy and prognosis.