Caveolin-1 was discovered as a major substrate for v-Src, but the effect of its tyrosine phosphorylation has not been known. We generated a specific antibody (PY14) to caveolin-1 phosphorylated at tyrosine 14 and studied the significance of the modification. By Western blotting of lysates of v-Src-expressing cells, PY14 recognized not only a 22-kDa band (the position of nonphosphorylated caveolin-1) but bands at 23-24 and 25 kDa. Bands of slower mobility were diminished by dephosphorylation and were also observed for mutant caveolin-1 lacking tyrosine 14. By immunofluorescence microscopy, PY14 did not label normal cells but detected large dots in v-Src-expressing cells. Immunoelectron microscopy revealed that the dots corresponded to aggregated caveolae and/or vesicles of various sizes; besides, the label was observed in intramembrane particle-free areas in the plasma membrane, which appeared to have been formed by fusion of flattened caveolae. A positive reaction with PY14 was found in normal cells after vanadate or pervanadate treatment; it occurred mainly at 22 kDa by Western blotting and was not seen as large dots by immunofluorescence microscopy. Detergent solubility, oligomerization, and association with caveolin-2 were observed similarly for caveolin-1 in normal and v-Src-expressing cells. The results indicate that phosphorylation of caveolin-1 in v-Src-expressing cells occurs at multiple residues and induces flattening, aggregation, and fusion of caveolae and/or caveolae-derived vesicles.