Kallikrein 6 is a serine protease expressed abundantly in normal adult human and rodent CNS, and therein is regulated by injury. In the case of CNS demyelinating disease, K6 expression in CNS occurs additionally in perivascular and parenchymal inflammatory cells suggesting a role in pathogenesis. Herein we describe two unique transcripts that occur within the human and mouse K6 genes that differ in their 5′-untranslated regions. These transcripts have identical translation initiation sites in exon 3, are expressed in a tissue-specific fashion and are differentially regulated in response to CNS injury. While the human and mouse 5′-transcripts differ in sequence they are identical in genomic organization and tissue-specific expression. The most 5′-transcript, designated transcript 1, includes exon 1–7, and was detectable in all CNS regions, but not in any non-CNS tissues examined (spleen, thymus, liver, kidney, pancreas, submandibular gland and peripheral nerve). In contrast, transcript 2 lacks exon 1, but contains a unique sequence at the 5′-end of exon 2, designated exon 2A. Transcript 2 was expressed both in CNS and in each peripheral tissue. In a murine model of human CNS demyelinating inflammatory disease induced by Theiler's picornovirus, mouse K6 transcript 1 was up-regulated in brain and spinal cord at acute and more chronic phases of CNS inflammation and demyelination, while overall transcript 2 expression was not significantly altered. However, in isolated splenocyte cultures, transcript 2 was up-regulated two-fold by cellular activation. Tissue-specific expression patterns and differential regulation in CNS disease indicates that each K6 5′-transcript is probably regulated by unique promoter elements and may serve as a molecular target to treat inflammatory demyelinating disease.