Human class I major histocompatibility antigens (HLA-A, -B and -C) are integral membrane protein heterodimers, which are anchored in the membrane via a stretch of hydrophobic amino acids near the carboxyl terminus of the heavy chain. It has previously been shown that a mutagenized cell line secretes a water soluble form of the HLA-A2 antigen, due to a pattern of RNA splicing that removes exon 5 (encoding the transmembrane hydrophobic amino acids) from mature, HLA-A2--encoding transcripts. The present study was undertaken to assess whether a similar process might be operative in nonmutagenized cells. It is shown that water soluble class I molecules (primarily HLA-A24) are secreted by the T leukemic cell line HPB-ALL, and that alternative splicing removes exon 5 from a fraction of HLA-A24--encoding transcripts. It is further shown that class I molecules are secreted, possibly in an allele-specific fashion, from a variety of tumor cells and normal cells. The possible relationship between these findings and previous reports of HLA-A and -B antigens in human serum is discussed.