Human immunodeficiency virus (HIV) spends a significant part of the viral life cycle as a latent provirus integrated into the host genome. Activation of latent HIV‐1 requires mitogenic stimulation of the cell, which increases basal viral transcription, and the HIV‐1 tat protein. As tat itself dramatically increases HIV‐1 gene expression, it too is presumably regulated in the latent state, and may also be activated by mitogenic stimulation. We show here that depletion of protein kinase C (PKC), which is essential to the stimulation of T cells by several mitogens, dramatically reduces HIV‐1 transactivation without affecting synthesis of tat protein. Transactivation in PKC‐depleted cells can be restored by transfection with a PKC expression vector. The requirement for PKC in trans‐activation does not involve the PMA‐responsive enhancer elements responsible for the effect of mitogens on basal transcription. Our results indicate that PKC regulates the process of HIV‐1 transactivation, suggesting a key role for the mitogenic induction of trans‐activation in the transition of HIV from latency to productive growth.