The α3(V) chain is poorly characterized among type V collagen chains. Pro-α3(V) collagen is expressed in newly synthesized bone as well as in the superficial fascia of developing muscle. Present study examined the expression in a mouse model of wound healing. Real-time reverse transcriptase polymerase chain reaction and in situ hybridization revealed transient expression of pro-α3(V) chain at a lower level than other fibrillar collagen genes after injury. Immunohistochemistry showed a similar expression pattern in the injured skin. In addition, electron microscopy showed that pro-α3(V) chain was localized in the amorphous nonfibrillar region, but not in fine or dense fibrils. The pro-α3(V) chain co-localized with heparan sulfate, which appeared in the skin after injury and might bind via an acidic segment of the pro-α3(V) chain. The matrix containing the pro-α3(V) chain may therefore be needed for the initiation of wound healing.