Vomeronasal organs of female Wistar rats were exposed with sprayed urine preparations of male Wistar rats prior to sacrifice. Exposure to crude urine and ultrafiltrated urine preparation (<5000 Da) induced significant Fos expression, which is correlated with cellular activity, in the mitral/tufted cell layer of the accessory olfactory bulb (AOB), while exposure to the remaining substances after ultrafiltration (>5000 Da) and control salt solution did not. Exposure to urine preparation treated with papain induced expression of Fos-immunoreactive cells in the rostral region of the AOB, but did not induce such expression in the caudal region. Exposure to urine preparation treated with pronase induced urine-specific Fos immunoreactivity neither in the rostral nor in the caudal region. These results suggest that at least two different peptides carrying pheromonal activities are contained in male Wistar rat urine.