Mitogenic anti‐CD28 antibody stimulates all peripheral T cells to proliferate in the absence of TCR ligation, providing an exception to the two‐signal requirement of T cell responses. This antibody preferentially recognizes a mobilized signaling‐competent form of CD28, normally induced following TCR ligation, thus providing a unique non‐physiological tool to dissect CD28‐specific signals leading to T cell proliferation. The protein kinase C (PKC)θ–NF‐κB pathway has recently been shown to integrate TCR‐ and CD28‐derived signals in co‐stimulation. We now demonstrate that this pathway is activated by mitogenic anti‐CD28 antibody stimulation. In contrast to conventional anti‐CD28 antibody, mitogenic anti‐CD28 antibody induced activation of phospholipase Cγ and Ca²⁺ flux in peripheral rat T cells despite no or low levels of inducible tyrosine phosphorylation of TCRζ chain, TCRζ‐associated protein of 70 kDa (ZAP‐70) or linker for activation of T cells (LAT)—critical components of the TCR signaling machinery. Nevertheless, PKCθ kinase activity in vitro was increased following mitogenic anti‐CD28 antibody stimulation, as was membrane association of both PKCθ and Bcl10. As downstream targets of PKCθ activation, NF‐κB components translocated to the nucleus at levels comparable to those after TCR–CD28 co‐stimulation. NF‐κB translocation was diminished by PKCθ inhibition, as was induction of the NF‐κB/AP‐1 responsive activation marker CD69. We propose that co‐stimulation is a sequential process in which appropriate TCR engagement is required to mobilize CD28 into a signaling‐competent form which then activates the PKCθ–NF‐κB pathway necessary for IL‐2 production and proliferation.