Protein kinase C (PKC) is a signal transduction protein that has been proposed to mediate rapid responses to steroid hormones. Previously, we have shown aldosterone directly activates PKC while 17-estradiol activates PKC and PKC, however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKC and PKC involved in binding of aldosterone and 17-estradiol, GST-fusion recombinant PKC and PKC mutants were used to perform in vitro binding assays with [³H]-aldosterone and [³H]-17-estradiol. 17-Estradiol bound both PKC and PKC but failed to bind PKC mutants lacking C2 domain. Similarly, aldosterone bound only PKC and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17-estradiol were between 0.5–1.0 nM. Aldosterone and 17-estradiol competed for binding to PKC, suggesting they share the same binding site. Phorbol 12,13-dibutirate did not compete with hormone binding; furthermore they have an additive effect on PKC activity. EC₅₀ for activation of PKC and PKC by aldosterone and 17-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKC and PKC undergo autophosphorylation with an EC₅₀ in the 0.5–1.0 nM range. 17-estradiol activated PKC and PKC in estrogen receptor positive and negative breast cancer cells (MCF-7 and HCC-38 respectively) suggesting estrogen receptor expression is not required for 17-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKC and PKC by steroid hormones and the molecular mechanisms involved.