A versatile negative-staining ribonuclease zymogram is described. The method has several advantages as it combines, by means of different staining procedures, high resolving power, sensitivity, and specificity with a rapid, reproducible, and simultaneous analysis of purity of ribonuclease samples on the same polyacrylamide gel. Activity bands can be visualized at any time during the incubation process without staining of the gel. This allows the choice of different staining procedures after incubation. Using poly(C) as substrate less than 1 pg of bovine pancreatic ribonuclease A was detected in less than 2 h after the electrophoretic run. An additional advantage with respect to other methods is that no refrigeration is needed during electrophoresis. © 1994 Academic Press, Inc. All rights reserved.