The goal of this study was to produce milligram quantities of pure, catalytically active, endotoxin-free recombinant neurolysin (rNln) in standard laboratory conditions for use as a research tool. To this end, we transformed E. coli cells with a plasmid construct for polyhistidine-tagged rNln, selected a high-expressing clone and determined the optimal time-point for translation of rNln. rNln was purified to homogeneity from the soluble pool of the cell lysate using Ni-NTA affinity and size-exclusion chromatography, followed by removal of endotoxins. Using this protocol ∼3 mg pure, catalytically active and nearly endotoxin-free (≈0.003 EU/μg protein) rNln was reproducibly obtained from 1 l of culture. Lack of cytotoxicity of rNln preparation was documented in cultured mouse cells, whereas stability in whole mouse blood. Intraperitonealy administered rNln in mice reached the systemic circulation in intact and enzymatically active form with Tmax of 1 h and T1/2 of ∼30 min. Administration of rNln (2 and 10 mg/kg) did not alter arterial blood pressure, heart rate, body temperature and blood glucose levels in mice. These studies demonstrate that the rNln preparation is suitable for cell culture and in vivo studies and can serve as a research tool to investigate the (patho)physiological function of this peptidase.