Abstract A rapidly growing weanling male Wistar rat was labeled by 18O 2 administration. After cessation of labeling, granulation tissue was induced by dorsal subcutaneous implantation of closed stainless steel mesh cylinders. These were removed at times varying from 6 to 21 days after implant, over a period of 41 days after cessation of labeling. Collagen hydroxy- l-proline (OHP) from skin and granulation tissue was isolated, purified, and derivatized for mass spectrometry (MS). MS analysis showed that 18O 2 labels rat skin collagen OHP in vivo in the hydroxy position. Unlike previous results from l-[ 3H]proline labeling studies, the granulation tissue in this experiment contained only 6–17% of the average (during implantation) skin isotope levels and did not vary significantly over time. We conclude that the majority of collagen deposited in granulation tissue is derived from local proteolysis of preformed collagen and efficient recycling of the amino acid substrate for de novo synthesis. The previously synthesized molecules or fragments that were translocated to the granulation tissue as identified by the 6–17% isotope transfer may reflect minimal structural reutilization but are more likely to be fragments of collagen undergoing proteolysis.