The purpose of the present study was to investigate smoking abstinence effects and the dissipation of tolerance (reactivity to nicotine) under controlled laboratory conditions. Seventeen male and female regular smokers were tested first in a session following ad libitum smoking and then in an additional five sessions over the course of 11 days during which they abstained from smoking. A metered dose of nicotine was administered via intranasal spray to ensure standard exposure, and pre- and post-dosing measures of heart rate, blood pressure, cortisol, galvanic skin response (GSR), craving, and several DSM-IV withdrawal symptoms (anxiety, irritability, restlessness, difficulty concentrating, and appetite) were collected. Prior to the nicotine test dose during deprivation sessions, heart rate and systolic blood pressure evinced elements of both an ‘offset abstinence pattern’ (deflection in a direction opposite to that produced by smoking) and a ‘transient abstinence pattern’ (deflection followed by a subsequent return); for cortisol, an offset pattern was observed, whereas for GSR and craving, a transient pattern was found. With respect to loss of tolerance, heart rate reactivity was found to increase significantly after 2 days' abstinence from nicotine, and the increase was sustained in subsequent sessions. Cortisol reactivity revealed more gradual dissipation, with significant differences evident only after 9 days of abstinence. These findings extend research on nicotine abstinence effects and on the dissipation of tolerance to nicotine deprivation intervals of nearly 2 weeks and confirm prior observations of variability across different response systems.