As peroxisomes possess some of the integral enzymes for cholesterol biosynthesis, the role of these organelles in cholesterol formation was studied in dermal fibroblasts with three types of peroxisomal defect: group I, characterized by the absence of intact peroxisomes (neonatal adrenoleukodystrophy, cerebrohepatorenal syndrome of Zellweger); group II, showing impaired activity of a single peroxisomal enzyme (X-linked adrenoleukodystrophy, adrenomyeloneuropathy); and group III, defective in more than one peroxisomal enzyme (rhizomelic chondrodysplasia punctata). Cells were incubated with three different radioactive precursors, namely [14C]-octanoate, [14C]-acetate, and [3H]-mevalonate, and incorporation of these radiolabels into cholesterol was determined. All fibroblasts with peroxisomal defects were able to form cholesterol at concentrations comparable or higher than those in controls dependent on the radioactive substrate. Binding properties (KD) and bmax values) of LDL to fibroblasts with peroxisomal defects and downregulation of intracellular cholesterol biosynthesis were similar to those found in fibroblasts from normolipidaemic controls, but different to those observed in LDL-receptor negative fibroblasts. As our studies revealed that cholesterol biosynthesis is not impaired in fibroblasts from patients with metabolic disorders of peroxisomal origin, we conclude that peroxisomes play little or no role in the pathway of cholesterol synthesis beyond mevalonate. In earlier steps of the cholesterol synthesis pathway, peroxisomal and mitochondrial defects in parallel may alter cholesterol synthesis indirectly.