The technology for the large-scale production of therapeutic recombinant proteins remains a challenge in the biopharmaceutical industry. In this study, we reported a nontransgenic approach to producing a large quantity of human nerve growth factor beta (hNGF-beta) in rabbit milk by employing a recombinant adenoviral expression system. After directly instilling hNGF-beta recombinant adenoviruses into rabbit mammary glands, a polypeptide with a molecular weight of 13.2 kDa was detected in rabbit milk. The maximal expression level of hNGF-beta reached 346 mu g/ml. The biological activity of recombinant hNGF-beta was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. Our data suggest that instilling recombinant adenovirus directly into the mammary gland of mammals is an efficient approach to producing a large quantity of hNGF-beta.