Liver mitochondrial P450c27, encoded by the CYP27 gene,can catalyse the 25-hydroxylation of vitamin D3 and the 27-hydroxylation of sterols. To facilitate the study of this enzyme in cell culture systems, we engineered a fusion protein consisting of P450c27 coupled to its electron-transport accessory proteins, ferredoxin and ferredoxin reductase, and assessed its enzyme activity by measuring the C-25 and C-27 (side-chain) hydroxylation of 1α-hydroxyvitamin D3 (1α-OH-D3). When incubated with 1α-OH-D3, COS-1 cells transfected with a vector expressing the fusion protein produced 1α,25-(OH)2D3 and 1α,27-(OH)2D3 about four times more efficiently than did cells transfected with three individual components of the fusion. When incubated with the natural substrate, vitamin D3, the efficiency of hydroxylation was lower than that for 1α-OH-D3 but still 1.7-fold higher for the fusion protein than for its individual components. The fusion protein was also ableto reproduce qualitatively and quantitatively the activity shown by P450c27 in liver cells in situ. The P450c27-ferredoxin reductase-ferredoxin fusion construct represents a valuable tool for establishing the substrate specificity of this liver cytochrome and for evaluating its potential for activating pro-drug analogues of vitamin D.