Methadone in vitro inhibited biogenic amine uptake into synaptosomes isolated from rat whole brain; the IC50 for inhibition of serotonin uptake was between 0.1 and 1 micron, and for norepinephrine uptake, 10 micron when the serotonin or norepinephrine concentration was 0.05 micron. Methadone in vitro also inhibited uptake of norepinephrine into rat brain synaptic vesicles to the same extent that it inhibited synaptosomal norepinephrine uptake. In all cases, all cases, morphine in vitro was much less effective in blocking uptake. However, in vivo acute or chronic administration of methadone to rats failed to cause inhibition of serotonin or norepinephrine uptakes in synaptosomes isolated from the brains of treated rats, nor was inhibition of vesicular norepinephrine uptake produced. Amine uptake in situ also was unaffected by methadone administration, as evidenced by normal incorporation of intracisternally administered 3H-serotonin into synaptic endings. These data show that inhibition of synaptosomal and vesicular biogenic amine uptake by methadone in vitro is probably not a major determinant of the alterations in biogenic amine turnover caused by methadone administration. Thus, results obtained with platelets and adrenomedullary chromaffin vesicles, in which methadone administration does cause effective inhibition of amine uptake in vivo, cannot be extended to the central nervous system.