In the development of cytotoxic T lymphocyte (CTL)-mediated immunotherapy, the identification of CTL epitopes is of crucial importance. Binding of a peptide to major histocompatibility complex (MHC) class I molecules is one of the prerequisites for its function as a CTL epitope. We describe the technique, validation, and application of a simple cellular assay, intended for the screening of peptides for binding, that can be applied to any human leukocyte antigen (HLA) allele. Reconstitution of peptides in MHC class I molecules after elution by acid treatment was previously shown to be possible in specially engineered cell lines expressing only one type of MHC class I, and was applied for the HLA-A*0201 allele. We now report the optimal conditions for application of this type of binding assay to the HLA-A*0301 allele. The adaptations that were necessary to make the technique operational for HLA-A*0301 are shown in detail. These consisted of lowering the pH during acid treatment to 2.9 and lengthening the duration of elution to 90 s. Furthermore, immediate aspiration of eluted peptides appeared to be essential for this allele. We found also that the use of Epstein-Barr virus (EBV)-transformed B cell lines (B-LCL) yields results similar to those of the use of cell lines expressing only one specific MHC class I allele. Homozygosity for the desired HLA allele improves the sensitivity of the assay, but heterozygous cells can also be employed. Finally, we applied this technique to a search for HLA-A*0301 binding peptides derived from carcinoembryonic antigen (CEA). Of a set of 34 CEA-specific peptides that fit with a specified HLA-A*0301-binding motif, we identified a set of six peptides with high binding affinity to this allele. These peptides can be regarded as potential CTL epitopes.