Repeated, intermittent treatment of rats with amphetamine results in a sensitization of locomotor and stereotyped behaviors that is accompanied by an enhancement in stimulus-induced dopamine release. The effects of repeated treatment with amphetamine on the phosphorylation state of neuromodulin and synapsin I, proteins involved in neurotransmitter release, were investigated. Rats were injected with 2.5 mg/kg AMPH, twice a week for 5 weeks (intermittent treatment). One week after the last injection, a challenge dose of 2.5 mg/kg AMPH was given 30 min before sacrifice. We previously reported an increase in neuromodulin phosphorylation with this sensitization paradigm. Site 3-phospho-synapsin I, site 1-phospho-synapsin I and phosphoser41-neuromodulin were detected with phosphorylation state-specific antibodies. Acute treatment with amphetamine did not increase the state of synapsin phosphorylation at either site 1 or site 3, but both site 1-phospho-synapsin I and site 3-phospho-synapsin I were increased (38% and 34%, respectively) after repeated, intermittent amphetamine. Immunoreactivity for phosphoser41-neuromodulin was increased by acute amphetamine. Site 3-phospho-synapsin I, site 1-phospho-synapsin I and phosphoser41-neuromodulin were also measured in striatum from rats receiving a different regimen in which amphetamine is given in escalating doses for 4 weeks. With this regimen, behavioral sensitization and enhanced dopamine release are exhibited in rats withdrawn 4 weeks, but not 3 days, after pretreatment. Small but significant increases in site 3-phospho-synapsin I and phosphoser41-neuromodulin were found in rats withdrawn 4 weeks from the escalating dose regimen, but not in those withdrawn 3 days. The increase in the phosphorylation state of synapsin I and neuromodulin reflect changes in the presynaptic signal transduction pathways which could play a role in the behavioral sensitization and contribute to the enhanced dopamine release reported in amphetamine-sensitized rats.