Abnormal production of superoxide (O(2)(-)) contributes to hypertension, in part because of its effects on the kidney. The thick ascending limb absorbs 20% to 30% of the filtered load of NaCl. O(2)(-) stimulates NaCl absorption by the thick ascending limb by enhancing Na(+)/K(+)/2Cl(-) cotransporter activity; however, the signaling mechanism is unknown. We hypothesized that O(2)(-) stimulates NaCl absorption by activating protein kinase C (PKC). To test this, we measured the effect of O(2)(-) on: (1) Cl(-) absorption in the presence and absence of PKC inhibitors, (2) total PKC activity, and (3) activation of specific PKC isoforms. Isolated perfused medullary thick ascending limbs were exposed to O(2)(-) generated by xanthine oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L). O(2)(-) increased Cl(-) absorption by 42% (from 76.2+/-3.6 to 108.2+/-11.9 pmol/min per millimeter; n=5; P<0.05). After treatment with the general PKC inhibitor staurosporine (10 nmol/L), O(2)(-) did not stimulate Cl(-) absorption (Delta-5.7+/-8.6%; n=6). In thick ascending limb suspensions, O(2)(-) increased total PKC activity by 33% (from 66+/-11 to 88+/-12 mU/mg protein; n=5; P<0.05) and increased PKC-alpha and PKC-delta activity by 1.75- and 0.37-fold, respectively. The PKC-alpha/beta-selective inhibitor Gö976 (100 nmol/L) blocked the ability of O(2)(-) to stimulate Cl(-) absorption by isolated perfused medullary thick ascending limbs (Delta4.5+/-15.0%; n=5). The role of PKC-delta could not be studied because of cell necrosis caused by the selective inhibitor rottlerin. We conclude that PKC-alpha is required for O(2)(-)-stimulated NaCl absorption in the thick ascending limb.