Hemolytic activity was identified in the saliva of Amblyomma americanum (L.) when red blood cells from sheep were incubated with tick saliva in the presence of phosphatidylcholine and sodium deoxycholate. The hemolytic activity was destroyed by boiling or treating with trypsin. The hemolytic activity in tick saliva was calcium-dependent, and inhibited by a phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine. Phosphatidylserine could replace phosphatidylcholine in the hemolytic assays but phosphatidylethanolamine and phosphatidylinositol were ineffective. Size exclusion chromatography of tick saliva revealed one peak of hemolytic activity, which correlated with the activity of tick salivary phospholipase A2, both having a molecular weight approximately 55,000 daltons. These results suggest that the hemolytic activity in tick saliva results from salivary phospholipase A2. The hemolytic activity in tick saliva may play a role in lysing host red blood cells, thus facilitating the tick digestive process.