Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.