Src plays an important role in cell proliferation, differentiation, adhesion, and migration. Altered Src activity has been strongly implicated in the development, growth, progression, and metastasis of human cancers. We have analysed the change and regulation of Src upon cell detachment in anoikis-resistant human lung adenocarcinoma cells and compared with that of relatively normal and anoikis-sensitive epithelial cells. We found that Src activity was increased in the anoikis-resistant lung tumor cells when they were detached and cultured in suspension. The detachment-induced Src activation in the tumor cells compensates for the loss of cell survival signals caused by disruption of cell--matrix interactions and contributes to anoikis resistance of the tumor cells. Pyk2, rather than PI 3K/Akt or Erk, appears to be the key downstream effecter of Src in mediating the cell survival signals. The increased Src activity is mainly due to the phosphorylation of Tyr-419, rather than the dephosphorylation of Tyr-530 of Src protein. PDGFR, not FAK or EGFR, appears to be the upstream protein tyrosine kinase responsible for the detachment-induced Src activation in the lung tumor cells. The increased Src activity upon cell detachment may contribute to the metastasis potential of malignant tumors.