Cholesteryl esters with various chain lengths of fatty acid, radioactive (C2-C18:1) and fluorescent (pyrene butanoic and decanoic acid, P4 and P10, respectively) were synthesized and their hydrolysis was investigated in lymphoid cell lines from normal subjects and from Wolman's disease patients. The comparison of their hydrolysis showed that three cholesterol esterases were present in normal lymphoid cell lines: the first, active at pH 4.0, hydrolysed preferentially cholesteryl esters of acyl chain length more than 8 carbons, and P10-cholesteryl ester. This acid cholesterol esterase, strongly inhibited by SH-blocking agents and resistant to E600, was severely deficient in Wolman lymphoid cell lines and corresponded to acid lysosomal lipase. The second and the third cholesterol esterases, active at pH 6.0 and 8.0, respectively, hydrolysed shorter-chain derivatives: the pH 8.0 enzyme was specific for short-chain derivatives (cholesteryl acetate, butyrate and P4), whereas the pH 6.0 activity showed a broader specificity, since it hydrolysed all the cholesteryl esters, with a maximum of activity on cholesteryl acetate and butyrate. The pH 6.0 and 8.0 enzymes were heat-labile, inhibited by E600, resistant to SH-blocking agents and not deficient in Wolman lymphoid cell lines. The hypothetical physiological role of these enzymes is discussed.