Cell-free, Ca2+-dependent phospholipase A2 activity (PLA2) was measured in human synovial fluid of patients with various kinds of arthritis using [1-14C] oleate-labelled autoclaved Escherichia coli as substrate. PLA2 activity at pH 7.0 and with 5 mM added Ca2+ was stimulated and then inhibited in a dose-dependent fashion by NaCl; maximal stimulation of 8.8 fold was found at 150 mM Na+. Similar effects were obtained with K+, Li+ and Ru+. In the absence of added Na+, PLA2 activity was maximal with 25 mM Ca2+ (145 nmols/hr/mg), but in the presence of 150 mM Na+, activity was maximal with 4 mM Ca2+ (415 nmols/hr/mg). PLA2 activity was optimal between pH 6.5-8.0 in presence of 150 mM Na+1 and 4 mM Ca2+. There was no significant difference between PLA2 activity in synovial fluids from rheumatoid and other types of arthritis. Neutral active, Ca2+-dependent PLA2 activity in acid extracts of human platelets, plasma, polymorphonuclear leukocytes and synovial fluid varied in response to added Na+. In presence of 150 mM added Na+ and 5 mM Ca2+, PLA2 activity in human synovial fluid was inhibited by all multivalent cations tested. In the absence of Na+, Cu2+ and Mg2+ stimulated PLA2 activity in a dose dependent fashion; whereas, Fe2+, Fe3+ and Al3+ were inhibitory. The extent of stimulation by Mg2+ was inversely related to the concentration of added Ca2+.