The present study was designed to test the hypothesis that preferential nuzzling of unfamiliar conspecific odors by male opossums is vomeronasally mediated. Opossums were presented with vials containing their own odors (Own) and vials with odors from unfamiliar conspecifics (Novel) both before and after (Expt. 1) or only after (Expt. 2) vomeronasal nerve (VNNX) or sham (Sham) surgery. Nuzzling duration was greater for Novel before (Expt. 1) and after (Expts. 1 and 2) surgery both in VNNX and Sham animals, indicating that a functional vomeronasal system is not necessary for the differential nuzzling of novel odors. In contrast, differential scent marking of Novel was lost following VNNX, but not following sham surgery. Since VNNX lesions disrupt differential marking behavior but, not differential nuzzling, it is possible that different components of conspecific odors guide these two chemosensory behaviors and that these components may be detected by different sensory systems. Vomeronasal nerve lesions, confirmed histologically, resulted in the loss of the beta-NADPH-dependent reaction product from the accessory olfactory bulbs (AOB). The pattern of staining with 4 lectins, soybean agglutinin (SBA), Phosphocarpus tetragonolobus (PCT), Vicia villosa agglutinin (VVA), and Griffonia simplicifolia lectin I-isolectin B4 (GS I-B4) remained unchanged in the AOB following vomeronasal nerve lesions, although the size of the AOB appeared to decrease in the VNNX animals.