Amino acid imaging is increasingly being used for assessment of brain tumor malignancy, extent of disease, and prognosis. This study explores the relationship between proliferative activity, amino acid transport, and glucose metabolism in three glioma cell lines (U87, Hs683, C6) at different phases of growth in culture. Growth phase was characterized by direct cell counting, proliferation index determined by flow cytometry, and [(3)H]thymidine (TdR) accumulation, and was compared with the uptake of two non-metabolized amino acids ([(14)C]aminocyclopentane carboxylic acid (ACPC) and [(14)C]aminoisobutyric acid (AIB)), and [(18)F]fluorodeoxyglucose (FDG). Highly significant relationships between cell number (density), proliferation index, and TdR accumulation rate were observed in all cell lines ( r>0.99). Influx ( K(1)) of both ACPC and AIB was directly related to cell density, and inversely related to the proliferation index and TdR accumulation in all cell lines. The volume of distribution ( V(d)) for ACPC and AIB was lowest during rapid growth and highest during the near-plateau growth phase in all cell lines. FDG accumulation in Hs683 and C6 cells was unaffected by proliferation rate, growth phase, and cell density, whereas FDG accumulation was correlated with TdR accumulation, growth phase, and cell density in U87 cells. This study demonstrates that proliferation rate and glucose metabolism are not necessarily co-related in all glioma cell lines. The values of K(1) and V(d) for ACPC and AIB under different growth conditions suggest that these tumor cell lines can up-regulate amino acid transporters in their cell membranes when their growth conditions become adverse and less than optimal.