Microsatellite instability is due to defects in the family of DNA repair genes, primarily hMLH1 and hMSH2, which can be detected by immunohistochemical staining. However, it is unclear whether immunohistochemical staining can accurately predict microsatellite instability status. We sought here to evaluate the sensitivity, specificity, and predictive values of immunostaining for the expression of the DNA mismatch-repair genes hMLH1 or hMSH2 in predicting microsatellite instability in ovarian carcinoma. Tissue microarrays with specimens from 322 women with primary ovarian carcinoma were stained with antibodies to hMLH1 and hMSH2; cases in which either hMLH1 or hMSH2 were negative were analyzed for microsatellite instability with the five-marker panel recommended by the National Cancer Institute (BAT26, BAT25, D5S346, D2S123, and D17S250). Microsatellite instability was also analyzed in another 19 cases selected at random in which both hMLH1 and hMSH2 were positive. Tumors with instability at two or more of the five NCI markers were considered to have a high level of microsatellite instability; tumors showing instability at only one marker were considered microsatellite instability-low; and tumors in which no markers exhibited microsatellite instability were considered microsatellite stable. We found that negative staining for hMLH1 protein (five cases) or hMSH2 protein (two cases) was associated with high level of microsatellite instability. The sensitivity and specificity of immunohistochemical staining for hMLH1 were 62 and 100% and those of hMSH2 alone were 25 and 100%. Combining loss of expression of both hMLH1 and hMSH2 led to sensitivity, specificity, and positive and negative predictive values of 87, 100, 100, and 95%. These results suggest that use of a two-molecule panel (hMLH1 and hMSH2) can accurately determine the microsatellite instability status of patients with ovarian cancer.