Incubation of quiescent Nakano mouse lens epithelial cells with sodium orthovanadate resulted in time- and concentration-dependent stimulation of protein tyrosine phosphorylation levels in the cells. Protein tyrosine phosphorylation in the 27,000 g pellet showed a 100% stimulation by vanadate. However, upon detergent solubilization, 30% activation of basal endogenous tyrosine phosphorylation was observed but no additional increase was obtained with vanadate. Protein phosphotyrosine phosphatase activity was found in both pellet and cytosolic fractions of the cell. Vanadate inhibited these activities with an IC50 of 57 microM in the cytosolic fraction and 3 microM in the pellet. These data suggest that vanadate increases phosphotyrosine protein levels in these cells by inhibition of a membrane-associated tyrosine-specific phosphatase rather than by activation of protein tyrosine kinases. These data correlate well with the vanadate stimulation of DNA synthesis in these cells, thus indicating a role for phosphotyrosine proteins in regulation of cell division in these cells.