The seed coats of S. spinosa (prickly sida, Malvaceae) become impermeable to water during seed development on the mother plant. After the seeds have dehydrated during the final maturation stages, piercing of seed coats is necessary to induce imbibition of water and germination. Onset of impermeability occurs during seed coat browning, well in advance of seed dehydration. I. Marbach and A.M. Mayer (1975, Plant Physiol. 56, 93-96) implicated polyphenol oxidase (PO; EC 1.10.3.1) as catechol oxidase in the formation of insoluble polymers during development of coat impermeability in a wild strain of pea (Pisum elatius) seeds. We found, however, that peroxidase (EC 1.11.1.7), not PO, is instrumental in the development of water-impermeable seed coats in prickly sida. We isolated coats and embryos from seeds harvested at several stages of development. Highest peroxidase activity of coat extracts correlated well with the developmental stages of maximum conversion of soluble phenolics to insoluble lignin polymers. Although seed extracts oxidized dihydroxyphenylalanine, this activity was eliminated by catalase, indicating that the oxidation of phenolics in the coat is catalyzed by peroxidase rather than PO. Histochemical localization of peroxidase was strongest in the palisade layer; both the level and time of appearance of activity was proportional to the spectrophotometric assays of seed-coat extracts. The presence of peroxidase and the absence of PO in the seed coat were also confirmed with immunocytochemistry. Our results support the view that peroxidase is involved in the polymerization of soluble phenolics to insoluble lignin polymers during development of prickly sida seed coats, causing the formation of a water-impermeable barrier prior to seed dehydration. As dehydration proceeds, the chalazal area finally becomes impermeable resulting in the hard mature seeds of prickly sida.