Spectrophotometric heparin assays which are based on the catalytic effect of heparin on either the inactivation of thrombin or that of factor Xa by antithrombin III, were adapted for use in a laboratory batch analyzer. Optimal conditions were determined for assays using the chromogenic substrates Chromozym-Th and S-2238 with thrombin, and S-2222 with factor Xa. Inactivation of the clotting enzyme by antithrombin III was stopped by addition of chromogenic substrate. Assays thus obtained appeared to be applicable in a wider range of heparin concentrations and were less dependent on plasma antithrombin III concentration that known manual spectrophotometric methods. The best results were obtained with the methods based on thrombin inactivation and applying a logarithmic reference curve.