Chylomicrons labeled with [3H]arachidonic and [14C]linoleic acid were incubated with bovine milk lipoprotein lipase or rat postheparin plasma, containing both lipoprotein lipase and hepatic lipase. During incubation with bovine lipoprotein lipase, [3H]arachidonic acid was released from chylomicron triacylglycerols at a slower rate than [14C]linoleic acid. Only small amounts of [14C]linoleic acid were found as 1,2(2,3)-diacylglycerols, whereas a transient accumulation as [14C]monoacylglycerols was observed. In contrast, significantly more [3H]arachidonic acid was found as 1,2(2,3)-diacylglycerols than as monoacylglycerols at all time intervals investigated. The initial pattern of triacylglycerol hydrolysis by postheparin plasma was similar to that of bovine lipoprotein lipase. However, in contrast to the results obtained with bovine lipoprotein lipase, little [3H]1,2(2,3)-diacylglycerol accumulated. The addition of antiserum to hepatic lipase increased the amount of 3H found in 1,2(2,3)-diacylglycerols and inhibited the formation of free [3H]arachidonic acid. The antiserum also caused a significant inhibition of the hydrolysis of [3H]-but not of [14C]triacylglycerol. With regard to chylomicron phospholipids, the rate of hydrolysis of [14C]linoleoyl phosphatidylcholine with milk lipoprotein lipase was twofold higher than that of the [3H]arachidonyl phosphatidylcholine. However, the hepatic lipase of postheparin plasma had similar activity towards the two phosphatidylcholine species. Postheparin plasma rapidly hydrolyzed chylomicron 3H-labeled and 14C-labeled phosphatidylethanolamine to the same degree, and lipoprotein lipase similarly hydrolyzed 3H-labeled and 14C-labeled phosphatidylethanolamine at approximately equal rates. Antiserum to hepatic lipase inhibited the postheparin plasma hydrolysis of phosphatidylethanolamine and 3H-labeled phosphatidylcholine by about 60%, but the 14C-labeled phosphatidylcholine by only 27%.(ABSTRACT TRUNCATED AT 250 WORDS)