Calcitriol (1,25-dihydroxyvitamin D3, 1,25(OH)2D3) plays a pivotal role in maintaining calcium and phosphate homeostasis. Aside from that, it supports the native and attenuates the acquired immune system and has positive effects on cell growth, differentiation and the prevention of carcinogenesis. The goal of this study was to detect possible differences in the expression of the calcitriol-degrading enzyme 24-hydroxylase (24-OHase) between malignant and benign ovarian cell lines and tissue. The analyses were based on real-time PCR, nested touchdown PCR and Western blot. When compared to benign granulose GLZ cells, the malignant HGL5 cells showed a significantly higher 24-OHase expression at the protein level (p<0.01). In the malignant ovarian tissue, the expression was significantly higher in RNA (p<0.001), but lower at the protein level (p<0.01). An increased 24-OHase expression could indicate a decrease in available calcitriol.