Prolylhydroxyproline (Pro-Hyp), which is derived from collagen hydrolysate, has been shown to be beneficial for skin and joint health. However, little is known about the distribution of Pro-Hyp in these tissues. In the present study, we investigated the biodistribution of orally administered [(14)C]Pro-Hyp in rats. Whole-body autoradiography at 30 min after administration of [(14)C]Pro-Hyp showed that radioactivity is widely distributed in tissues including skin and articular cartilage, with the highest level of radioactivity observed in the gastric and intestinal walls. Incorporation of radioactivity into cells known to respond to Pro-Hyp such as dermal fibroblasts, synovial cells, chondrocytes, osteoblasts, and osteoclasts was observed. The chemical form of [(14)C]Pro-Hyp-derived radioactivity detected in the tissues was investigated by thin layer chromatography. The radioactive constituents in cartilage extract were two proline-modified peptides (56%), intact Pro-Hyp (5%), and two nonpeptide metabolites (28%). Similar results were obtained for skin and bone marrow. Plasma analysis at 3 to 30 min post-dose suggested that the majority of Pro-Hyp is modified in its proline residue by a first-pass effect without peptide bond hydrolysis. In conclusion, we demonstrated that Pro-Hyp is partly distributed in observed tissues including skin and cartilage in its intact form, which might be responsible for its biological functions.