We analyzed the protein composition of human aqueous humor. Samples were obtained by paracentesis from 25 human eyes (age range 64-92 years) at elective cataract surgery, and from 20 age-matched post-mortem eyes within 1.5 to 18 hr after death. Individual samples were assayed for total protein, and the polypeptides were separated by qualitative SDS-PAGE into high-, medium- and low-molecular-weight ranges and then silver-stained. The clinical samples showed a remarkable consistency in the total protein values (mean +/- SEM: 12.4 +/- 2.0 mg per 100 ml) and no detectable variations in the profiles of the silver-stained proteins. Twelve major protein fractions, with apparent molecular weights of 140, 80 (doublet), 67, 60 (doublet), 35, 27, 25, 17, 14.6 and 9 kDa, were present. A preliminary analysis showed that the 17 kDa band contained a molecule resembling basic fibroblast growth factor. Two additional samples of aqueous humor from patients whose blood/aqueous barrier was compromised during paracentesis showed a quantitative and qualitative increase in the polypeptides that were present. Compared with the samples of aqueous humor obtained at surgery, the post-mortem samples exhibited a greater variability in total protein content (56.1 +/- 11.6 mg per 100 ml) and an increased number of high- and low-molecular-weight protein fractions. In view of wide differences in the clinical parameters, including ocular and systemic medications, systemic illness, surgical premedications, anesthesia and total serum protein values, the similarity in the protein profiles of the carefully drawn surgical samples is most remarkable. Our results indicate that, in patients who underwent elective cataract surgery, the levels of major proteins in human aqueous humor are not affected by wide individual variations in the clinical parameters. We attribute this finding to the care taken in the collection of aqueous humor samples.