The binding specificities of a panel of mouse monoclonal antibodies (MAbs) to human nerve growth factor (hNGF) were determined by epitope mapping using chimeric and point mutants of NGF. Subsequently, the MAbs were used to probe NGF structure-function relationships. Six MAbs, which recognize distinct or partially overlapping regions of hNGF, were evaluated for their ability to block the binding of hNGF to the TrkA and p75 NGF receptors in various in vitro assays, which included blocking of TrkA autophosphorylation and blocking of NGF-dependent survival of dorsal root ganglion sensory neurons. Three MAbs (911,912,938) were potent blockers of all activities. Potent blocking of p75 binding occurs only with MAb 909, which recognizes an NGF region identified by mutagenesis as important for NGF-p75 binding. These results are consistent with recently proposed models of binding regions involved in NGF-TrkA and NGF-p75 interactions generated through mutagenic analysis and structure determination of the NGF-TrkA complex. These studies provide insight to the epitope specificities and potency of MAbs that would be useful for physiological NGF blocking studies.