1. The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling-activated Cl- current (I-Cl,I-swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole-cell patch clamp technique. I-Cl,I-swell was activated by reducing the extracellular osmolality by either 12.5% (mild hypotonicity) or 25% (strong hypotonicity). 2. The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited I-Cl,I-swell with IC50 values of, respectively, 9.2 +/- 0.2, 61.4 +/- 1.7 and 62.9 +/- 1.3 mu M. Tyrphostin Al, a tyrphostin analogue with little effect on PTK activity and daidzein, an inactive genistein analogue, were without effect on I-Cl,I-swell. 3. The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 mu M) and dephostatin (20 mu M) potentiated I-Cl,I-swell activated by mild hypotonicity by 47 +/- 9 and 69 +/- 15%, respectively. 4. Intracellular perfusion with GTP gamma S (100 mu M) transiently activated a Cl- current with an identical biophysical and pharmacological profile to I-Cl,I-swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity-induced cell shrinkage completely inhibited the GTP gamma S-activated Cl- current. 5. Intracellular perfusion with GDP beta S (1 mM) caused a time-dependent inhibition of I-Cl,I-swell, which was more pronounced when the current was activated by mild hypotonicity. 6. Our results demonstrate that the activity of endothelial swelling-activated Cl- channels is dependent on tyrosine phosphorylation and suggest that a proteins regulate the sensitivity to cell swelling.