The activated form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), plays an important role in the immune system. Indeed, receptors for 1,25(OH)(2)D(3) are found on most immune cells, and 1alpha-hydroxylase, the enzyme responsible for final activation of vitamin D(3), is expressed by monocytes/macrophages, resulting in secretion of 1,25(OH)(2)D(3) after immune stimulation. We have previously shown that in murine peritoneal macrophages 1alpha-hydroxylase is highly regulated by immune signals such as IFNgamma and LPS. In the present study we made use of two different knock-out mouse models with disruptions in two key transcription factors in the IFNgamma-signalling cascade (STAT1alpha and IRF1), to evaluate their role in the regulation of 1alpha-hydroxylase. This was performed by culturing peritoneal macrophages from these knock-out mice in the presence of IFNgamma and LPS, and evaluating the impact of the absence of the respective transcription factors on 1alpha-hydroxylase mRNA expression by real-time RT-PCR. In addition also the mRNA expression profiles of the essential transcription factors STAT1alpha, IRF1 and C/EBPbeta were investigated. The data confirm a crucial role for STAT1alpha as well as for C/EBPbeta in the regulation of 1alpha-hydroxylase in monocytes.