The instability of short repetitive sequences in tumor DNA can result from defective repair of replication errors due to mutations in any of several genes required for mismatch repair. Understanding this repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the mismatch repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific mismatch repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The repair defects were independent of whether the signal for strand specificity, a nick, was 5' or 3' to the mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective mismatch repair and the suggestion that diminuition in mismatch repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of repair-proficient and repair-deficient cell lines for future studies of mismatch repair.