Antigenic peptides are presented to cytotoxic T lymphocytes by heterodimers of MHC class I molecules and β₂ ‐microglobulin. Peptides are generated in the cytosol and translocated into the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP). Optimal binding of peptides to class I molecules is facilitated by the physical association between class I heterodimers and TAP. This association is mediated largely by the glycoprotein tapasin. Analysis of tapasin function has relied on a mutant cell line, .220, which is defective in tapasin expression and antigen presentation. We have investigated the genetic basis of these defects. In .220 cells, Tapasin transcripts lack exon two. This is caused by a single nucleotide substitution, disrupting the 5 ′ splice site of the second intron. A tapasin protein is produced in .220 cells, but has a truncated signal peptide and lacks the N‐terminal 49 amino acids encoded by full‐length transcripts. Nonetheless, this truncated form is translocated into the ER and interacts with TAP. As a result of alternative splicing, transcripts lacking exon two are also present in wild‐type cells, although no truncated protein was detected. Additionally we describe a polymorphism in the Tapasin gene, with two alleles encoding arginine or threonine at peptide position 240.