Endogenous pp60c-src from Y79 unsynchronized human retinoblastoma cells is phosphorylated at an additional N-terminal serine residue relative to unsynchronized fibroblast pp60c-src. We confirmed that the novel phosphorylation site is Ser 75, which is the same as that phosphorylated in overexpressed human pp60c-src during NIH3T3 cell mitosis. We also showed that the Ser 75 phosphorylation pattern correlates with cell rounding in 14 various unsynchronized human tumor cell lines. Especially, pp60c-src from Lu135 having a spherical morphology similar to that of Y79 is phosphorylated as high as Y79 pp60c-src. Mitotic spherical cells of the epithelial-like HepG2 express pp60c-src phosphorylated on Ser 75. On the other hand, Y79 pp60c-src is phosphorylated on Ser 75 throughout the cell cycle. These data suggest that this phosphorylation may be important in spherical cell morphology.