Canine splenic hemangiosarcoma (HSA) is a spontaneous tumor with high metastatic potential. Despite surgical excision, most dogs die within 2 months of diagnosis as a result of widespread visceral metastasis. This study was designed to determine the efficacy of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) when used in combination with splenectomy and systemic chemotherapy for the treatment of HSA in the dog. Thirty-two dogs with HSA and without gross evidence of metastases were treated with splenectomy, stratified by clinical stage, and randomized to receive doxorubicin/cyclophosphamide chemotherapy and either L-MTP-PE immunotherapy or lipid equivalent (placebo liposomes). Dogs were subsequently followed to determine disease-free survival and overall survival times. The effects of L-MTP-PE on serum tumor necrosis factor-alpha and interleukin 6 activity were assessed on a small subset of dogs. Dogs receiving L-MTP-PE had significantly prolonged disease-free survival (P = 0.037) and overall survival (P = 0.029) compared with dogs receiving placebo. Dogs with clinical stage I disease had significantly prolonged disease-free survival (P = 0. 026) and overall survival (P = 0.017) compared with dogs with clinical stage II disease. Dogs receiving L-MTP-PE had significantly greater serum tumor necrosis factor-alpha (P < 0.001) and interleukin 6 (P = 0.007) activities compared with placebo-treated dogs. L-MTP-PE has significant antimetastatic activity in highly malignant, spontaneously occurring, splenic HSA in the dog. Canine HSA may have potential as a large animal model for additional investigation of antimetastatic chemoimmunotherapy.