Translocation from the cytoplasm to the nucleus is required for the regulation of gene expression by transcription factors of the nuclear factor kappa B (NF-kappaB) family. The p65:p50 NF-kappaB heterodimer that predominates in many cell types can undergo stimulated movement, following degradation of the IkappaB inhibitor, as well as shuttling in the absence of stimulation with IkappaB bound. Disruption of the dynactin complex and knockdown of endogenous dynein were used to investigate the nuclear translocation requirements for stimulated and shuttling movement of NF-kappaB. A differential dependence of these two modes of transport on the dynein molecular motor and dynactin was found. NF-kappaB used active dynein-dependent transport following stimulation while translocation during shuttling was mediated by a dynein-independent pathway that could be potentiated by dynactin disruption, consistent with a process of facilitated diffusion. Nuclear translocation and activation of NF-kappaB-dependent gene expression showed a dependence on endogenous dynein in a variety of cell types and in response to diverse activating stimuli, suggesting that dynein-dependent transport of NF-kappaB may be a conserved mechanism in the NF-kappaB activation pathway and could represent a potential point of regulation.