Regulation of swelling-activated Cl⁻ current (I_Cl,swell) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates I_Cl,swell and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). I_Cl,swell evoked by hyposmotic swelling was increased 181 ± 17% by 100 μM genistein, and the genistein-induced current was blocked by the selective I_Cl,swell blocker tamoxifen (10 μM). Block of Src with PP2 (10 μM) stimulated tamoxifen-sensitive I_Cl,swell by 234 ± 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 μM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited I_Cl,swell and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited I_Cl,swell. Several lines of evidence argue that the PP2-stimulated current was I_Cl,swell: 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl⁻ gradients, and 4) the current reversed near the Cl⁻ equilibrium potential. To rule out contributions of other currents, Cd²⁺ (0.2 mM) and Ba²⁺ (1 mM) were added to the bath. Surprisingly, Cd²⁺ suppressed the decay of I_Cl,swell, and Cd²⁺ plus Ba²⁺ eliminated time-dependent currents between −100 and +100 mV. Nevertheless, these divalent ions did not eliminate I_Cl,swell or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls I_Cl,swell, and regulation of I_Cl,swell by the Src and EGFR kinase families of PTK is antagonistic.