A subset of hereditary and sporadic colorectal carcinomas is defined by microsatellite instability (MSI), but the spectra of gene mutations have not been characterized extensively. Thirty-nine hereditary nonpolyposis colorectal cancer syndrome carcinomas (HNPCCa) and 57 sporadic right-sided colonic carcinomas (SRSCCa) were evaluated. Of HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa ( P < 0.000001), but instability tended to be more widespread in SRSCCa ( P = 0.08). Absence of nuclear hMSH2 mismatch repair gene product by immunohistochemistry was associated with germline hMSH2 mutation ( P = 0.0007). The prevalence of K- ras proto-oncogene mutations was similar in HNPCCa and SRSCCa (30% (11/37) and 30% (16/54)), but no HNPCCa from patients with germline hMSH2 mutation had codon 13 mutation ( P = 0.02), and two other HNPCCa had multiple K- ras mutations attributable to subclones. 18q allelic deletion and p53 gene product overexpression were inversely related to MSI ( P = 0.0004 and P = 0.0001, respectively). Frameshift mutation of the transforming growth factor β type II receptor gene was frequent in all MSI-positive cancers (85%, 46/54), but mutation of the E2F-4 transcription factor gene was more common in HNPCCa of patients with germline hMSH2mutation than in those with germline hMLH1 mutation (100% (8/8) versus 40% (2/5), P = 0.04), and mutation of the Bax proapoptotic gene was more frequent in HNPCCa than in MSI-positive SRSCCa (55% (17/31) versus 13% (2/15), P = 0.01). The most common combination of mutations occurred in only 23% (8/35) of evaluable MSI-positive cancers. Our findings suggest that the accumulation of specific genetic alterations in MSI-positive colorectal cancers is markedly heterogeneous, because the occurrence of some mutations (eg, ras, E2F-4, and Bax genes), but not others (eg, transforming growth factor β type II receptor gene), depends on the underlying basis of the mismatch repair deficiency. This genetic heterogeneity may contribute to the heterogeneous clinical and pathological features of MSI-positive cancers.