Aging in rodents, monkeys, and man is correlated with a reduction in dopamine transporter (DAT) ligand binding and DAT function. Using Western blot techniques, we investigated whether the source of these age-related changes in DAT was correlated with decreases in DAT protein levels in the striatum, substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) of 6, 18, and 24-month-old male Fischer 344 rats. The relative levels of tyrosine hydroxylase (TH) were also determined in each region. In the striatum, we also assessed [3H]-DA uptake and DAT plasma membrane expression using a membrane-impermeant biotin analog in crude synaptosomes prepared from these age groups. There was no significant age-related difference in DAT immunoreactivity per total protein or per total TH in striatum, NAc, SN, or VTA. Significant age-related changes in TH were only seen in the VTA of the 24-month-old rats (approximately 60% decrease). However, [3H]-DA uptake and DAT protein recovered in the biotinylated fraction in 24-month-old rats were significantly decreased (approximately 30%) compared to 6-month-old animals in the striatal synaptosomes. These data suggest that age-related decreases in striatal DAT function and ligand binding are related to a decrease in plasma membrane expression of DAT and not a decrease in the steady-state levels of DAT protein or loss of dopaminergic neuropil.