The mechanism related to the decrease in the number of fibroblasts in the dermis during aging and the decrease in the biosynthetic capacity of existing fibroblasts to produce collagen is not known. Since prolidase [E. C. 3. 4. 13. 9] has been found to play an important role in the recycling of proline for collagen synthesis and cell growth, we performed comparative studies on prolidase activity and extracellular collagen content in in vitro aged human skin fibroblasts. We found that during aging of human skin fibroblasts there is a correlated decrease of prolidase activity and extracellular collagen content. The correlation is bifunctional. Decrease in prolidase activity contributes to decrease of extracellular collagen content, and removal of extracellular collagen form cultured fibroblasts contributes to a decrease of fibroblast prolidase activity. Using plates coated with different ECM proteins, it was found that extracellular type I collagen in cultured fibroblasts was the most potent activator of intracellular prolidase activity. Our results demonstrate that during in vitro aging of human skin fibroblasts, the prolidase activity of fibroblasts decreases. This phenomenon may be related to the reduced number of fibroblasts and decrease of extracellular collagen content in in vitro aging of the cells.