Recent studies in our laboratory have demonstrated the presence of specific binding sites for [3H](+)-amphetamine in crude membrane preparations derived from rat brain. In this report we have further characterized the specific binding of [3H](+)-amphetamine in various subcellular fractions of rat brain and demonstrate a greater than five-fold enrichment in the crude synaptosomal (P2) fraction compared to a crude membrane preparation. Specific [3H](+)-amphetamine binding in crude synaptosomal membranes in saturable and stereospecific with an apparent dissociation constant, Kd, of 2.8 ± 0.5 μM and an estimated maximum number of binding sites, Bmax, of 60.4 ± 8.4 pmoles/mg protein derived by Scatchard or Klotz analysis of binding data using filtration assays. Centrifugation assays yield a similar Kd though the apparent Bmax is higher. In addition specific [3H](+)-amphetamine binding is: rapidly reversible, temperature sensitive, labile to preincubation in Tris buffer, inhibited by sodium ions and unevenly distributed in various brain regions. Specific [3H](+)-amphetamine binding sites are found almost exclusively in the rat central nervous system (the brainstem, hypothalamus, and striatum exhibiting relatively high levels of binding), whereas peripheral tissues such as liver, kidney and heart have very low to undetectable levels of specific binding.