The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the α2u-globulin (α2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified α2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel α2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats. This novel α2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated denovo sequences which provided additional evidence for the presence of α2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to α2u. The present investigation confirms the presence of α2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat. Copyright © 2010 John Wiley & Sons, Ltd.