AbstractNur77 orphan steroid receptor and its family member Nor‐1 are required for apoptosis of developing T cells. In thymocytes, signals from the TCR complex induce Nur77 and Nor‐1 expression followed by translocation from the nucleus to mitochondria. Nur77 and Nor‐1 associate with Bcl‐2 in the mitochondria, resulting in a conformation change that exposes the Bcl‐2 BH3 domain, a presumed pro‐apoptotic molecule of Bcl‐2. As Nur77 and Nor‐1 are heavily phosphorylated, we examined the requirement of Nur77 and Nor‐1 phosphorylation in mitochondria translocation and Bcl‐2 BH3 exposure. We found that HK434, a PKC agonist, in combination with calcium ionophore, can induce Nur77 and Nor‐1 phosphorylation, translocation, Bcl‐2 BH3 exposure and thymocyte apoptosis. Inhibitors of both classical and novel forms of PKC were able to block this process. In contrast, only the general but not classical PKC‐specific inhibitors were able to block the same process initiated by PMA, a commonly used PKC agonist. These data demonstrate a differential activation of PKC isoforms by PMA and HK434 in thymocytes, and show the importance of PKC in mitochondria translocation of Nur77/Nor‐1 and Bcl‐2 conformation change during TCR‐induced thymocyte apoptosis.