Nerve growth factor (NGF) is a protein essential for the survival and normal function of sympathetic neurons. Two-site immunoassays have been developed over the past decade in several laboratories and used to estimate its endogenous concentrations in a variety of effector tissues. However, levels appear restricted to a narrow range, display only a poor correlation with innervation density, and show obvious inter- and intralaboratory variations, the origins of which are unclear. This led us to examine alternative extraction procedures for NGF before quantification. In particular, we have found treatment of tissue extracts with high and low pH in the presence of detergent results in the detection of higher NGF concentrations in immunoassays using either polyclonal or commercially available monoclonal antibodies. These increases were tissue-specific (sciatic nerve, mesenteric arteries, and thoracic aorta > heart and brain > sympathetic ganglia > abdominal aorta) and as much as 10 times greater than the amounts detected by traditional procedures. The method should also prove useful for the assay of other members of the neurotrophin family when appropriate antibodies become available.