The subcellular distribution of prolyl hydroxylase was determined in rat fibroblasts and chondrocytes immunohistochemically. After fixation in 4 % paraformaldehyde fibroblasts were treated with a specific goat antiserum to rat prolyl hydroxylase followed by rabbit anti-goat IgG conjugated with horseradish peroxidase. After further fixation in glutaraldehyde the bound intracellular peroxidase activity was identified by a standard histochemical procedure. Electron microscopy of the final product showed that the antigen was located only on the membranes of the rough endoplasmic reticulum. From ultracentrifugation analysis of fibroblast homogenates 80 % of the prolyl hydroxylase activity could be located in the microsomal fraction. These observations suggest that not only collagen chain synthesis but also proline hydroxylation occurs on the membranes of the rough endoplasmic reticulum.