New generation topical hemostatic agents containing thrombin have been developed for use in surgical procedures when control of bleeding by conventional methods is either ineffective or impractical. The authors compared the safety, hemostatic efficacy, and handling characteristics of a thrombin-containing topical surgical hemostatic agent (a gelatin matrix) to a hemostatic gelatin sponge for treatment of parenchymal bleeding after liver biopsy. Fourteen dogs were enrolled in this prospective clinical study. Paired 1.5 cm × 1.5 cm and 0.5 cm deep liver biopsies were obtained via laparotomy for each dog. One bleeding liver biopsy lesion was treated with the gelatin matrix and the other with a gelatin sponge. The treated liver biopsy sites were compared for bleeding severity, time to hemostasis, cumulative blood loss, and hemostatic agent handling characteristics. Median time to hemostasis was significantly shorter (P = 0.034) and median cumulative blood loss was significantly lower (P = 0.033) for the lesions treated with the gelatin matrix than the gelatin sponge. Adverse reactions were not observed within the first 24 hr postoperatively. When used to control parenchymal bleeding from liver biopsy sites in the dog, the evaluated gelatin matrix was safe and more effective than the gelatin sponge.