H eat shock proteins (HSP) are components of the steroid receptor complex and are released into the cell cytosol after hormone binding. We tested whether HSPs released from steroid receptors mediate an increase in calcineurin phosphatase activity by steroid hormones. Aldosterone increased calcineurin activity in microdissected rat cortical collecting ducts (CCD) and connecting tubules, but not in proximal tubules, medullary thick ascending limb, or outer medullary collecting ducts. In contrast, 5 microM dexamethasone increased calcineurin activity in both CCD and proximal tubules. Aldosterone increased CCD calcineurin activity after 30 min and this response was blocked by spironolactone, but not by actinomycin D. An antibody recognizing HSP-56 did not change basal calcineurin activity, but completely blocked the stimulation of calcineurin by aldosterone. Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid receptor complex, also blocked the aldosterone response, whereas HSP-90 or HSP-70 increased calcineurin activity in permeabilized CCD. In summary, (a) aldosterone increases calcineurin activity in CCD through a transcription-independent process; (b) maneuvers inactivating HSP-56 or slowing HSP disassociation from the receptor complex blocks stimulation of calcineurin by steroid hormones; (c) HSP-90 and HSP-70 increase CCD calcineurin activity in the absence of steroid hormone. We conclude that HSPs released from transformed steroid receptors can stimulate calcineurin activity through a transcription-independent pathway.