A procedure for enzymatic determination of serum triglycerides [Clin. Chem. 19, 476 (1973)] has been adapted for use in continuous-flow analysis (Technicon AutoAnalyzer). A very simple manifold is used; serum is incubated at 37 °C with the lipase and α-chymotrypsin in potassium phosphate buffer (0.1 mol/liter, pH 7, containing 1.50 g of bovine serum albumin per liter). The liberated glycerol is dialyzed against the complete glycerol reagent. The change in absorbance at 340 nm resulting from oxidation of NADH is proportional to the dialyzed glycerol. The same manifold can be used to determine preformed glycerol if the hydrolyzing enzymes are omitted. The lipase used need not be extensively purified because the dialyzer removes coloring material and contaminating enzymes that would interfere with the components of the glycerol reagent in the manual procedure. The hydrolysis is complete, as shown by the use of equivalent glycerol standards. No prior treatment of the samples is necessary. Assays are run at 60 per hour in the AutoAnalyzer I, 80 per hour in the AutoAnalyzer II. Results with both instruments for 150 samples correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure.