The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However, no easily applicable approach for CTL epitope identification has been developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector. Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide. The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2Ld-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131. The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.