Purpose: To assess vitreous metabolism by measuring Cpropeptide levels of type II procollagen (pCOL-II-C) and hyaluronan levels in the vitreous and in the vitreous fluid after vitrectomy for macular hole. Methods: We obtained 1-ml vitreous samples during vitrectomy from34 patients (34 eyes)with a macular hole (age range 50-77 years, mean 64 years). After vitrectomy, we performed fluid-air exchange in six eyes because of unresolved macular holes and collected 4-ml fluid samples. Gel-filtration high-performance liquid chromatography (HPLC) was used to determine the molecular weight of pCOLII-C in the samples. The pCOL-II-C level was measured by sandwich enzyme immunoassay and hyaluronan by sandwich binding protein assay. Results: HPLC showed that pCOL-IIC in the vitreous samples corresponded to purified pCOL-II-C from cartilage. The vitreous pCOL-II-C level (4.7±0.3 ng/ml)-was similar to reported synovial fluid levels. In six eyes that underwent fluid-air exchange, pCOL-II-C in the fluid samples remained at a level similar to that in the vitreous samples, while hyaluronan levels in the fluid samples were significantly lower than in the vitreous samples. Conclusions: The molecular weight and concentrations of pCOL-II-C in the vitreous are similar to those in joint fluid. In patients with a macular hole, type II procollagen may be secreted persistently into the vitreous cavity before and after vitrectomy. Purpose: To assess vitreous metabolism by measuring Cpropeptide levels of type II procollagen (pCOL-II-C) and hyaluronan levels in the vitreous and in the vitreous fluid after vitrectomy for macular hole. Methods: We obtained 1-ml vitreous samples during vitrectomy from 34 patients (34 eyes) with a macular hole (age range 50-77 years, mean 64 years). After vitrectomy, we performed fluid-air exchange in six eyes because of unresolved macular holes and collected 4-ml fluid samples. The pCOL-II-C level was measured by a sandwich enzyme immunoassay and hyaluronan by sandwich binding protein assay. Results: HPLC showed that pCOL The vitreous pCOL-II-C level (4.7 ± 0.3 ng / ml) -was similar to reported synovial fluid levels. In six eyes that underwent fluid-air exchange, pCOL-II-C in the flui d samples remained at a level similar to that in the vitreous samples, while hyaluronan levels in the fluid samples were significantly lower than in the vitreous samples. Conclusions: The molecular weight and concentrations of pCOL-II-C in the vitreous are similar to those in joint fluid. In patients with a macular hole, type II procollagen may be secreted persistently into the vitreous cavity before and after vitrectomy.