The putative serine protease PRSS16 is abundantly expressed in the thymic cortex and the gene is encoded within the HLA I complex. Although its function is not yet defined, the very restricted expression points to a role in T-cell development in the thymus. In this study, we show that the PRSS16 mRNA is alternatively spliced to generate at least five transcripts. Apart from the full-length sequence, we found two other isoforms with all putative active site residues of the serine protease, suggesting that those variants may also be functional. Semi-quantitative analysis of the splice variants in different tissue samples revealed a strong correlation between the specific formation of alternatively spliced PRSS16 transcripts and the age and thymus pathology status of the donor. Newborn thymi express mostly the PRSS16-4 and -5 isoforms and lack the PRSS16-1 transcript, which appears around 2 years of age and stays until adulthood. Incidentally, thymi from myasthenia gravis (MG) patients with thymoma showed a marked decrease in the expression of the full-length PRSS16-1 and increased expression of the smaller isoforms. The data suggest a potential role of the PRSS16 isoforms in the postnatal morphogenesis of the thymus and in the thymus pathology related to MG.