In previous studies we have shown that rat adipocytes suspended in Matrigel and placed in primary culture migrate through the gel to form multicellular clusters over a 5- to 6-day period. In the present study, phosphorylation of the insulin-regulated 70-kDa ribosomal protein S6 kinase (p70S6k) was observed within 30 min of establishment of adipocytes in primary culture. Two inhibitors of the p70S6k signaling pathway, rapamycin and LY-294002, greatly reduced phosphorylation of p70S6k and organization of adipocytes into multicellular clusters. Of all the components of the cell culture medium, amino acids, and in particular a subset of neutral amino acids, were found to promote both phosphorylation of p70S6k and cluster formation. Lowering the concentrations of amino acids in the medium to levels approximating those in plasma of fasted rats decreased both phosphorylation of p70S6k and cluster formation. Furthermore, stimulation of p70S6k phosphorylation by amino acids was prevented by either rapamycin or LY-294002. These findings demonstrate that amino acids stimulate the p70S6k signaling pathway in adipocytes and imply a role for this pathway in multicellular clustering.