Previous studies have established that rat primary astrocyte cultures prepared from several brain regions of 1–4-d-old rats exhibit high- affinity, Na(+)-dependent and fluoxetine-sensitive serotonin (5-HT) uptake with a Km for 5-HT of 0.4 microM and a Ki for fluoxetine of 23 nM, which correspond to the characteristics for this transport for other brain preparations. However, it is not known whether astrocytes in situ show such uptake. We addressed this question by performing 3H-5- HT uptake experiments on cortical astrocytes, within 4 hr of isolating them from 6- and 21-d-old rats by the tissue-print technique. Quantitative autoradiography was combined with GFAP and neurofilament (NF) immunocytochemistry to distinguish astrocytic from neuronal 3H-5- HT uptake. In composition, the tissue-printed (TP) cells and processes were 60–70% GFAP (+) and 10–15% NF(+). 3H-5-HT uptake (0.3 microM 5-HT, 3.4 microCi/ml) in both tissue-printed GFAP(+) astrocytes and NF(+) structures was sensitive to 1 microM fluoxetine and was also Na+ dependent. More than 90% of TP astrocytes from 6- and 21-d-old rats and 100% of NF(+) structures from 21-d-old rats showed positive 3H-5-HT uptake (defined as > or = 31 grains/10(3) microns2). The highest level of uptake (> or = 191 grains/10(3) microns2) was never observed in TP astrocytes but was exhibited by about half of the NF(+) structures. In other experiments were found that 3H-5-HT uptake by 6-d-old TP astrocytes was comparable to uptake by postnatal age-matched primary cultured astrocytes that were grown in fetal bovine serum (FBS). However, primary cultured astrocytes grown in horse serum showed lower uptake than that observed with FBS, a finding similar to previous results in cultures where 3H-5-HT uptake was measured per milligram of cell protein. These results imply that high-affinity, Na(+)-dependent and fluoxetine-sensitive 5-HT uptake occurs in rat cortical astrocytes in situ.