In view of the proposed role of the non-specific lipid-transfer protein (nsL-TP; sterol carrier protein 2) in the metabolism of pulmonary surfactant lipids (Batenburg et al. (1994) Biochem. J. 298, 223-229), its subcellular localization was studied in the surfactant producing alveolar type II cells. It was shown by immuno-electron microscopy that nsL-TP colocalizes with the peroxisomal marker catalase. The peroxisomal localization of nsL-TP was confirmed by gradient fractionation of type II cell homogenates. As a peroxisomal marker acyl-CoA:dihydroxyacetone-phosphate acyltransferase was assayed. Given this subcellular localization, it is very unlikely that nsL-TP plays a role in the transfer of surfactant lipids from the endoplasmic reticulum to the lamellar bodies. These results strengthen the opinion that peroxisomes are involved in surfactant synthesis.