OBJECTIVE The present study was conducted to determine lysosomal phospholipase A2 (LPLA2) activity in the aqueous humor (AH) and to identify the possible sources of the LPLA2 found in the AH. METHODS To detect LPLA2 activity in pig AH and ocular tissues, liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine were used as substrates in an activity assay under acidic conditions. The reaction products were separated by thin-layer chromatography. To identify the LPLA2 in pig AH, the AH was analyzed by Western immunoblot analysis with an anti-LPLA2 antibody. Distribution of the LPLA2 in pig ocular tissues was studied by determining its activity in individual tissue extracts. RESULTS LPLA2 activity was detected in the AH obtained from pig eyes. Consistent with the known properties of LPLA2, the activity was heat-labile and undetectable at neutral pH. The immunoblot of pig AH showed the anti-LPLA2 antibody-reactive protein band. In addition, the specific activity of the enzyme, when normalized to volume, was higher in pig AH than in pig serum. Individual tissue extracts obtained from pig ocular tissues showed different specific activity of LPLA2. In particular, the extract prepared from the trabecular meshwork provided the highest specific activity. CONCLUSIONS The present findings suggest that the phospholipase A2 activity found in pig AH under acidic conditions is due to LPLA2 and that it originates from ocular tissues surrounding the anterior chamber as well as plasma.