An in vivo method has been developed for quantifying cavernosal endothelium-dependent relaxation. The method is based on the fact that relaxation of the smooth muscle around the sinusoids of the penile corpora cavernosa activates the erectile veno-occlusive mechanism, and the degree of veno-occlusion can be precisely quantified by the pharmacologic maintenance erectile flow (PMEF) method. Pharmacologic maintenance erectile flows are determined after intracavernosal infusion of the endothelium-dependent relaxant acetylcholine (ACh) and the endothelium-independent relaxant papaverine, and expressed as an acetylcholine/papaverine ratio (APR). Control rabbits showed no changes from the test procedures themselves. In 12 test rabbits, control PMEFs after approximately 10(-7) mol ACh or papaverine averaged 0.7 and 0.5 ml/minute, respectively; APR averaged 1.3. Endothelial injury of the corpus cavernosum was produced by intracavernosal injection of 100 micrograms (16 x 10(-8) mol) of the detergent CHAPS or 1 ml of Renografin-76. Within 1 hour of injection of either agent, PMEF(ACh) increased markedly to approximately 6, PMEF(pap) increased minimally to approximately 0.9, and APR increased to about 7. These values gradually decreased to normal limits at six weeks. Endothelial injury and recovery were confirmed by electron microscopy. Thus, reduced cavernosal response to ACh relative to papaverine was indicative of endothelial injury. The ACh/papaverine response ratio offers promise as a practical and reliable in vivo method for quantifying endothelial-dependent relaxation.