Perinatal exposure to nicotine has been shown to cause morphological and neurobehavioral abnormalities in developing brain. In the current study, neonatal rats were given an acute injection of nicotine (3 mg/kg) at 1, 3, 8, 10 or 15 days of age, and [3H]thymidine incorporation into DNA examined over the 30-min period after drug administration. Three brain/regions were used that differ in their timetables of cell maturation and in their concentrations of nicotinic receptors. Nicotine inhibited DNA synthesis in all brain regions but with a rank order of effect corresponding to the concentration of nicotinic receptors, namely midbrain + brainstem greater than or equal to cerebral cortex greater than cerebellum. Superimposed on this hierarchy, periods of rapid cell replication were more sensitive to nicotine, so that drug effects in the cerebellum, which develops last, became significant past the point at which nicotine no longer affected DNA synthesis in the other regions. The inhibitory effect of nicotine was also found in fetal brain on gestational day 20 after injection of nicotine to pregnant rats. Studies with adrenergic and ganglionic blocking agents and with 100% O2 indicated that autonomic and respiratory actions of nicotine, including ischemia, cardiac arrhythmias and hypoxia, could not solely account for the inhibition of DNA synthesis in neonatal brain. In contrast, injection of a small amount (2 micrograms) of nicotine directly into the central nervous system readily caused inhibition; the same small dose given systemically had no effect. These data suggest that nicotine damages the developing brain, in part, through direct actions on cell replication.