By the microsatellite assay, two types of genetic alterations, loss of heterozygosity (LOH) and replication error (RER), were examined using 7 dinucleotide repeat markers [D3S1317 (3p26); CI3-1169 (3p25); CI3-946 (3p25); D3S1255 (3p24.2-25); CI3-771 (3p21.3); CI3-1413 (3p14.1-14.3); and CI3-373 (3p13)] on the short arm of chromosome 3p as well as 3 markers [D2S123 (2p15-16), IFNA (9p22), and D16S408 (16q12.1-13)] on other chromosomes in 35 patients with esophageal squamous cell carcinoma. On 3p, LOH was detected in 34% (12 of 35) and RER was detected in 60% (21 of 35) at single or multiple loci. RER occurred at a similar frequency in all stages and did not correlate with clinicopathological characteristics. On the other hand, LOH at the 3p25 locus was more frequently detected in carcinomas with lymph node metastasis than in those without it (P < 0.05). The incidences of microsatellite alterations were low on the chromosomes other than 3p, except at D2S123, where the incidence of RER was 20%. These findings suggest that RER on 3p is an early event and that a tumor suppressor gene which is involved in the progression of esophageal squamous cell carcinoma may exist near the 3p25 locus.