1. Several methods are used to provide predictable and effective nicotine to experimental animals in scientific studies. Due to the expense and technical challenges of these methods, we sought suitable alternatives. Consequently, the purpose of the present study was to develop a reliable experimental nicotine protocol in rabbits that included either Habitrol(R) nicotine patches (Novartis Consumer Health Inc., Summit, NJ, USA) or nicotine nasal spray. 2. Administration of one of three doses of nicotine (2.5, 5, or 10 mg) was accomplished daily on 13 rabbits divided between either the patch or spray groups. Systemic nicotine and cotinine levels at 0 h, 15 min and 8 and 24 h were assayed. Data were analysed by a Fisher's protected least significant difference test at P = 0.05. 3. Rabbits treated with Habitrol(R) patches exhibited consistent and predictable systemic nicotine levels. The nicotine nasal spray produced an immediate dose-dependent response with no measurable nicotine serum levels at 8 h. 4. For nicotine administration in rabbits, nicotine patches are easy to administer and provide a nicotine serum level between 5 and 25 ng/mL, which is consistent with the average daily level found in a patient who smokes cigarettes.