Substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SIDS-PAGE) has become a popular procedure for the separation and identification of active fractions present in enzyme mixtures due to its relative simplicity. Procedures including high-molecular-mass substrates within the gel,,such as starch for identification of amylase activity, and protein substrates, including gelatin, casein, and collagen, for revealing protease activity, have been described. SIDS-PAGE separation under denaturing conditions is dependent on the molecular mass of the proteins and on the effective pore size of the gels, the last factor being affected by the inclusion of high-molecular-mass substrates into the polyacrylamide matrix. In order to quantify the effect of the addition of increasing concentrations of such substrates on protein migration, starch, gelatin, and casein were included in gels in which polyacrylamide concentration was kept constant. High-molecular-mass substrates decreased migration of proteins ranging from 6.5 to 205 kDa, although the migration pattern, and thereby the accuracy of the assignation of relative molecular masses to proteins separated on those gels, was practically unaffected. The substitution of glycine, as the carrying ion, by Tricine in denaturing electrophoresis buffer systems resulted in an improvement of the migration of proteins: in substrate-containing gels. Results suggested that zymograms including substrates remain a valuable procedure for the separation and the relative molecular mass assignation of active enzyme fractions.