The human tissue kallikrein family (KLK for protein; KLK for gene) includes 15 members. Twelve kallikreins, including KLK6, are concurrently upregulated in ovarian cancer. However, the mechanism of this phenomenon remains unclear. In this study, we measured KLK6 expression in a large series of ovarian tissue cytosols and examined possible mechanisms of KLK6 up-regulation in ovarian cancer. Using a newly developed enzyme-linked immunosorbent assay (ELISA) with two monoclonal antibodies, we quantified KLK6 expression in ovarian tissue cytosols, and confirmed the upregulation of KLK6 in ovarian cancer and its unfavourable prognostic value. We then examined KLK6 mRNA expression using reverse transcription-polymerase chain reaction and established its good concordance with KLK6 protein expression. This finding suggested that the KLK6 gene is under transcriptional regulation. We then scrutinised a few mechanisms that could explain KLK6 upregulation. The relative abundance of two KLK6 mRNA transcripts was studied; we found the same differential expression pattern in all samples, regardless of KLK6 levels. Genomic mutation screening of all exons and the 5'-flanking region of the KLK6 gene identified two linked single-nucleotide polymorphisms in the 5'-untranslated region, but neither correlated with KLK6 expression. Ovarian cell lines were separately treated with five steroid hormones. None of the treatments produced significant effects on KLK6 expression. We conclude that KLK6 is transcriptionally upregulated in ovarian cancer, but probably not through alternative mRNA transcript expression, genomic mutation, or steroid hormone induction.